m6A epitranscriptomic and epigenetic crosstalk in liver fibrosis: Special emphasis on DNA methylation and non-coding RNAs.

Liver fibrosis is a pathological process caused by a variety of chronic liver diseases. Currently, therapeutic options for liver fibrosis are very limited, highlighting the urgent need to explore new treatment approaches. Epigenetic modifications and epitranscriptomic modifications, as reversible regulatory mechanisms, are involved in the development of liver fibrosis. In recent years, researches in epitranscriptomics and epigenetics have opened new perspectives for understanding the pathogenesis of liver fibrosis. Exploring the epigenetic mechanisms of liver fibrosis may provide valuable insights into the development of new therapies for chronic liver diseases. This review primarily focus on the regulatory mechanisms of N6-methyladenosine (m6A) modification, non-coding RNA, and DNA methylation in organ fibrosis. It discusses the interactions between m6A modification and DNA methylation, as well as between m6A modification and non-coding RNA, providing a reference for understanding the interplay between epitranscriptomics and epigenetics.

Biclustering for Epi-Transcriptomic Co-functional Analysis.

Dynamic and reversible N6-methyladenosine (m6A) modifications are associated with many essential cellular functions as well as physiological and pathological phenomena. In-depth study of m6A co-functional patterns in epi-transcriptomic data may help to understand its complex regulatory mechanisms. In this chapter, we describe several biclustering mining algorithms for epi-transcriptomic data to discover potential co-functional patterns. The concepts and computational methods discussed in this chapter will be particularly useful for researchers working in related fields. We also aim to introduce new deep learning techniques into the field of co-functional analysis of epi-transcriptomic data.

Ocular A-to-I RNA editing signatures associated with SARS-CoV-2 infection.

Ophthalmic manifestations have recently been observed in acute and post-acute complications of COVID-19 caused by SARS-CoV-2 infection. Our precious study has shown that host RNA editing is linked to RNA viral infection, yet ocular adenosine to inosine (A-to-I) RNA editing during SARS-CoV-2 infection remains uninvestigated in COVID-19. Herein we used an epitranscriptomic pipeline to analyze 37 samples and investigate A-to-I editing associated with SARS-CoV-2 infection, in five ocular tissue types including the conjunctiva, limbus, cornea, sclera, and retinal organoids. Our results revealed dramatically altered A-to-I RNA editing across the five ocular tissues. Notably, the transcriptome-wide average level of RNA editing was increased in the cornea but generally decreased in the other four ocular tissues. Functional enrichment analysis showed that differential RNA editing (DRE) was mainly in genes related to ubiquitin-dependent protein catabolic process, transcriptional regulation, and RNA splicing. In addition to tissue-specific RNA editing found in each tissue, common RNA editing was observed across different tissues, especially in the innate antiviral immune gene MAVS and the E3 ubiquitin-protein ligase MDM2. Analysis in retinal organoids further revealed highly dynamic RNA editing alterations over time during SARS-CoV-2 infection. Our study thus suggested the potential role played by RNA editing in ophthalmic manifestations of COVID-19, and highlighted its potential transcriptome impact, especially on innate immunity.

Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA.

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Identification of m6A RNA methylation genes in Oryza sativa and expression profiling in response to different developmental and environmental stimuli.

Eukaryotic messenger RNAs (mRNAs) transcend their predominant function of protein encoding by incorporating auxiliary components that ultimately contribute to their processing, transportation, translation, and decay. In doing so, additional layers of modifications are incorporated in mRNAs at post-transcriptional stage. Among them, N6-methyladenosine (m6A) is the most frequently found mRNA modification that plays crucial roles in plant development and stress response. In the overall mechanism of m6A methylation, key proteins classified based on their functions such as writers, readers, and erasers dynamically add, read, and subtract methyl groups respectively to deliver relevant functions in response to external stimuli. In this study, we identified 30 m6A regulatory genes (9 writers, 5 erasers, and 16 readers) in rice that encode 53 proteins (13 writers, 7 erasers, and 33 readers) where segmental duplication was found in one writer and four reader gene pairs. Reproductive cells such as sperm, anther and panicle showed high levels of expression for most of the m6A regulatory genes. Notably, writers like OsMTA, OsMTD, and OsMTC showed varied responses in different stress and infection contexts, with initial upregulation in response to early exposure followed by downregulation later. OsALKBH9A, a noteworthy eraser, displayed varied expression in response to different stresses at different time intervals, but upregulation in certain infections. Reader genes like OsECT5, OsCPSF30-L3, and OsECT8 showed continuous upregulation in exertion of all kinds of stress relevant here. Conversely, other reader genes along with OsECT11 and OsCPSF30-L2 were observed to be consistently downregulated. The apparent correlation between the expression patterns of m6A regulatory genes and stress modulation pathways in this study underscores the need for additional research to unravel their intricate regulatory mechanisms that could ultimately contribute to the substantial development of enhanced stress tolerance in rice through mRNA modification.

Functional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection.

Epitranscriptomic RNA modifications play a crucial role in the posttranscriptional regulation of gene expression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic RNA and plays a pivotal role in RNA fate. RNA m6A modification is regulated by a group of cellular proteins, methyltransferases (writers) and demethylases (erasers), which add and remove the methyl group from adenosine, respectively. m6A modification is recognized by a group of cellular RNA-binding proteins (readers) that specifically bind to m6A-modified RNA, mediating effects on RNA stability, splicing, transport, and translation. The functional significance of m6A modification of viral and cellular RNA is an active area of virology research. In this review, we summarize and analyze the current literature on m6A modification of HIV-1 RNA, the multifaceted functions of m6A in regulating HIV-1 replication, and the role of viral RNA m6A modification in evading innate immune responses to infection. Furthermore, we briefly discuss the future directions and therapeutic implications of mechanistic studies of HIV-1 epitranscriptomic modifications.

Emerging roles of RNA ac4C modification and NAT10 in mammalian development and human diseases.

RNA ac4C modification is a novel and rare chemical modification observed in mRNA. Traditional biochemical studies had primarily associated ac4C modification with tRNA and rRNA until in 2018, Arango D et al. first reported the presence of ac4C modification on mRNA and demonstrated its critical role in mRNA stability and translation regulation. Furthermore, they established that the ac4C modification on mRNA is mediated by the classical N-acetyltransferase NAT10. Subsequent studies have underscored the essential implications of NAT10 and mRNA ac4C modification across both physiological and pathological regulatory processes. In this review, we aimed to explore the discovery history of RNA ac4C modification, its detection methods, and its regulatory mechanisms in disease and physiological development. We offer a forward-looking examination and discourse concerning the employment of RNA ac4C modification as a prospective therapeutic strategy across diverse diseases. Furthermore, we comprehensively summarize the functions and mechanisms of NAT10 in gene expression regulation and pathogenesis independent of RNA ac4C modification.

Corrigendum: Development and validation of a m6A -regulated prognostic signature in lung adenocarcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.947808.].

Synapse-Enriched m6A-Modified Malat1 Interacts with the Novel m6A Reader, DPYSL2, and Is Required for Fear-Extinction Memory.

The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.

Analysis of N6-methyladenosine-modified mRNAs in diabetic cataract.

BACKGROUND: Cataracts remain a prime reason for visual disturbance and blindness all over the world, despite the capacity for successful surgical replacement with artificial lenses. Diabetic cataract (DC), a metabolic complication, usually occurs at an earlier age and progresses faster than age-related cataracts. Evidence has linked N6-methyladenosine (m6A) to DC progression. However, there exists a lack of understanding regarding RNA m6A modifications and the role of m6A in DC pathogenesis. AIM: To elucidate the role played by altered m6A and differentially expressed mRNAs (DEmRNAs) in DC. METHODS: Anterior lens capsules were collected from the control subjects and patients with DC. M6A epitranscriptomic microarray was performed to investigate the altered m6A modifications and determine the DEmRNAs. Through Gene Ontology and pathway enrichment (Kyoto Encyclopedia of Genes and Genomes) analyses, the potential role played by dysregulated m6A modification was predicted. Real-time polymerase chain reaction was further carried out to identify the dysregulated expression of RNA methyltransferases, demethylases, and readers. RESULTS: Increased m6A abundance levels were found in the total mRNA of DC samples. Bioinformatics analysis predicted that ferroptosis pathways could be associated with m6A-modified mRNAs. The levels of five methylation-related genes-RBM15, WTAP, ALKBH5, FTO, and YTHDF1-were upregulated in DC samples. Upregulation of RBM15 expression was verified in SRA01/04 cells with high-glucose medium and in samples from DC patients. CONCLUSION: M6a mRNA modifications may be involved in DC progression via the ferroptosis pathway, rendering novel insights into therapeutic strategies for DC.

Alteration of the N6-methyladenosine methylation landscape in a mouse model of polycystic ovary syndrome.

OBJECTIVE: To explore the N6-methyladenosine (m6A) methylation abnormality of mRNAs and its potential roles in the mouse model of polycystic ovary syndrome (PCOS). METHODS: The mouse model of PCOS were induced by injecting dehydroepiandrosterone (DHEA), and confirmed by observing the morphological structures of ovarian follicles. Subsequently, m6A-tagged mRNAs were identified via m6A epitranscriptomic microarray and its potential functional pathways were predicted in KEGG database. The expression and modification levels of key mRNAs in the most enriched pathway were evaluated and compared using western blot and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR). RESULTS: Compared with the control group, 415 hypermethylated and downregulated mRNAs, 8 hypomethylated and upregulated mRNAs, and 14 hypermethylated and upregulated mRNAs were identified in the PCOS group (Fold change ≥ 1.5). Those mRNAs were mainly involved in insulin signaling pathway, type II diabetes mellitus, Fc epsilon RI signaling pathway, inositol phosphate metabolism, and GnRH secretion. In insulin signaling pathway, the expression levels of phosphorylated protein kinase B (p-AKT) were decreased, whereas that of upstream phosphorylated phosphatidylinositol 3-kinase (p-PI3K) were increased in PCOS group. Moreover, skeletal muscle and kidney-enriched inositol polyphosphate 5-phosphatease (SKIP), one of PIP3 phosphatases, was verified to be overexpressed, and Skip mRNAs were hypermethylated in PCOS group. CONCLUSION: The altered m6A modification of mRNAs might play a critical role in PCOS process. The PI3K/AKT pathway is inhibited in the mouse model of PCOS. Whether it is caused by the m6A modification of Skip mRNAs is worthy of further exploration.

Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells.

Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. We show that N6-methyladenosine (m6A) methylation of cell-cycle-related mRNAs ensures the proper cell-cycle progression of late-born retinal progenitor cells (RPCs), which are born toward the end of retinogenesis and have long cell-cycle length. Conditional deletion of Mettl14, which is required for depositing m6A, led to delayed cell-cycle exit of late-born RPCs but has no effect on retinal development prior to birth. m6A sequencing and single-cell transcriptomics revealed that mRNAs involved in elongating the cell cycle were highly enriched for m6A, which could target them for degradation and guarantee proper cell-cycle progression. In addition, we identified Zfp292 as a target of m6A and potent inhibitor of RPC cell-cycle progression.

Cancer metastasis under the magnifying glass of epigenetics and epitranscriptomics.

Most of the cancer-associated mortality and morbidity can be attributed to metastasis. The role of epigenetic and epitranscriptomic alterations in cancer origin and progression has been extensively demonstrated during the last years. Both regulations share similar mechanisms driven by DNA or RNA modifiers, namely writers, readers, and erasers; enzymes responsible of respectively introducing, recognizing, or removing the epigenetic or epitranscriptomic modifications. Epigenetic regulation is achieved by DNA methylation, histone modifications, non-coding RNAs, chromatin accessibility, and enhancer reprogramming. In parallel, regulation at RNA level, named epitranscriptomic, is driven by a wide diversity of chemical modifications in mostly all RNA molecules. These two-layer regulatory mechanisms are finely controlled in normal tissue, and dysregulations are associated with every hallmark of human cancer. In this review, we provide an overview of the current state of knowledge regarding epigenetic and epitranscriptomic alterations governing tumor metastasis, and compare pathways regulated at DNA or RNA levels to shed light on a possible epi-crosstalk in cancer metastasis. A deeper understanding on these mechanisms could have important clinical implications for the prevention of advanced malignancies and the management of the disseminated diseases. Additionally, as these epi-alterations can potentially be reversed by small molecules or inhibitors against epi-modifiers, novel therapeutic alternatives could be envisioned.

Epitranscriptomic profiling of N4-acetylcytidine-related RNA acetylation in the spinal dorsal horn of rat with cancer-induced bone pain.

Recently, epigenetics involved in the regulation of gene expression has become a research hotspot. This study evaluated N4-acetylcytidine (ac4c) RNA acetylation in the spinal dorsal horn (SDH) of rats with cancer-induced bone pain (CIBP). The ac4C-specific RIP sequencing and NAT10-specific RIP sequencing were performed to identify the differences in ac4C acetylation and gene expression in the SDH between CIBP and sham groups, the relationship with the acetylation-modifying enzyme NAT10, and association analysis was performed. By interfering with the NAT10 expression, the relationship between some up-regulated genes and ac4C acetylation in CIBP was verified. In this study, we demonstrated that bone cancer increases the levels of NAT10 and the overall acetylation, inducing differential ac4C patterns in the SDH of rats. Through verification experiments, it was found that ac4C acetylation of some genes is regulated by NAT10, and differential ac4C patterns in RNA determine the expression of this RNA. We exposed that some CIBP-related gene expression was altered in the SDH of rats, which was regulated by differentially expressed ac4C acetylation.

N6-methyladenosine mRNA methylation is important for the light response in soybean.

N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is the most prevalent and abundant modification in eukaryotic mRNA and posttranscriptionally modulates the transcriptome at almost all stages of mRNA metabolism. In plants, m6A is crucial for embryonic-phase growth, flowering time control, microspore generation and fruit maturation. However, the role of m6A in plant responses to light, the most important environmental stimulus, remains unexplored. Here, we profile the m6A transcriptome of Williams 82, a soybean cultivar, and reveal that m6A is highly conserved and plays an important role in the response to light stimuli in soybean. Similar to the case in Arabidopsis, m6A in soybean is enriched not only around the stop codon and within the 3'UTR but also around the start codon. Moreover, genes with methylation occurring in the 3'UTR have higher expression levels and are more prone to alternative splicing. The core genes in the light signaling pathway, GmSPA1a, GmPRR5e and GmBIC2b, undergo changes in methylation modification and transcription levels in response to light. KEGG pathway analysis revealed that differentially expressed genes with differential m6A peaks were involved in the "photosynthesis" and "circadian rhythm" pathways. Our results highlight the important role played by epitranscriptomic mRNA methylation in the light response in soybean and provide a solid basis for determining the functional role of light on RNA m6A modification in this plant.

Emerging Regulatory Mechanisms of N6-Methyladenosine Modification in Cancer Metastasis.

Cancer metastasis is the major cause of cancer-related deaths and accounts for poor therapeutic outcomes. A metastatic cascade is a series of complicated biological processes. N6-methyladenosine (m6A) is the most abundant and conserved epitranscriptomic modification in eukaryotic cells, which has great impacts on RNA production and metabolism, including RNA splicing, processing, degradation and translation. Accumulating evidence demonstrates that m6A plays a critical role in regulating cancer metastasis. However, there is a lack of studies that review the recent advances of m6A in cancer metastasis. Here, we systematically retrieved the functions and mechanisms of how the m6A axis regulates metastasis, and especially summarized the organ-specific liver, lung and brain metastasis mediated by m6A in various cancers. Moreover, we discussed the potential application of m6A modification in cancer diagnosis and therapy, as well as the present limitations and future perspectives of m6A in cancer metastasis. This review provides a comprehensive knowledge on the m6A-mediated regulation of gene expression, which is helpful to extensively understand the complexity of cancer metastasis from a new epitranscriptomic point of view and shed light on the developing novel strategies to anti-metastasis based on m6A alteration.

Self-attention enabled deep learning of dihydrouridine (D) modification on mRNAs unveiled a distinct sequence signature from tRNAs.

Dihydrouridine (D) is a modified pyrimidine nucleotide universally found in viral, prokaryotic, and eukaryotic species. It serves as a metabolic modulator for various pathological conditions, and its elevated levels in tumors are associated with a series of cancers. Precise identification of D sites on RNA is vital for understanding its biological function. A number of computational approaches have been developed for predicting D sites on tRNAs; however, none have considered mRNAs. We present here DPred, the first computational tool for predicting D on mRNAs in yeast from the primary RNA sequences. Built on a local self-attention layer and a convolutional neural network (CNN) layer, the proposed deep learning model outperformed classic machine learning approaches (random forest, support vector machines, etc.) and achieved reasonable accuracy and reliability with areas under the curve of 0.9166 and 0.9027 in jackknife cross-validation and on an independent testing dataset, respectively. Importantly, we showed that distinct sequence signatures are associated with the D sites on mRNAs and tRNAs, implying potentially different formation mechanisms and putative divergent functionality of this modification on the two types of RNA. DPred is available as a user-friendly Web server.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis.

OBJECTIVE: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. METHODS: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. RESULTS: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0]). CONCLUSIONS: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

The N6-methyladenosine RNA landscape in the aged mouse hippocampus.

The aged brain is associated with an inevitable decline in cognitive function and increased vulnerability to neurodegenerative disorders. Multiple molecular hallmarks have been associated with the aging nervous system through transcriptomics and proteomic studies. Recently, epitranscriptomic analysis has highlighted the role of RNA chemical modification in various biological processes. In particular, N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNAs, has been functionally linked to multiple aspects of RNA metabolism with the roles of m6A in processes such as learning and memory, leading to our current investigation of how the m6A-transcriptomic landscape is shaped during aging. Using the inbred C57BL/6 line, we compared the m6A-transcriptomic profiles from the hippocampi of young (3-month-old) and aged (20-month-old) mice. Methylated RNA immunoprecipitation (MeRIP)-sequencing analysis revealed hyper- and hypomethylation in 426 and 102 genes, respectively, in the aged hippocampus (fold change >1.5, false discovery rate <0.05). By correlating the methylation changes to their steady-state transcript levels in the RNA-Seq data, we found a significant concordance between m6A and transcript levels in both directions. Notably, the myelin regulator gene Gpr17 was downregulated in the aged hippocampus concomitant with reduced m6A levels in its 3'UTR. Using reporter constructs and mutagenesis analysis, we demonstrated that the putative m6A sites in the 3'UTR of Gpr17 are important for mRNA translation but not for regulating transcript stability. Overall, the positive correlation between m6A and the transcript expression levels indicates a co-transcriptional regulation of m6A with gene expression changes that occur in the aged mouse hippocampus.