Transcriptome-wide m6A methylation profile reveals tissue specific regulatory networks in switchgrass (Panicum virgatum L.) under cadmium stress.

The heavy metal cadmium (Cd), known for its high toxicity, poses a grave threat to human health through the food chain. N6-methyladenosine (m6A), the most abundant internal modification, regulates plant adaptation to various adversities, yet the panorama of m6A modifications in switchgrass under cadmium stress remains elusive. This study examines the physiological responses of switchgrass roots and shoots exposed to 50 µM CdCl2, alongside an overview of transcriptome-wide m6A methylation patterns. After cadmium treatment, methylation modifications are primarily enriched near stop codons and the 3'UTR region, with a negative correlation between m6A modification and gene expression levels. In shoots, approximately 58 % of DEGs with m6A modifications show upregulation in expression and decrease in m6A peaks, including zinc transporter 4-like (ZIP4). In roots, about 43 % of DEGs with m6A modifications exhibit downregulation in expression and increase in m6A peaks, such as the ABC transporter family member (ABCG25). We further validate the m6A enrichment, gene expression and mRNA stability of ZIP4 in response to Cd treatment. The results suggest that the negative correlation of m6A enrichment and gene expression is due to altered mRNA stability. Our study establishes an m6A regulatory network governing cadmium transport in switchgrass roots and shoots, offering new avenues for candidate gene manipulation in phytoremediation applications of heavy metal pollution.

Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures.

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.

Exposure to bisphenol A affects transcriptome-wide N6-methyladenine methylation in ovarian granulosa cells.

Bisphenol A (BPA) is an endocrine disruptor of potential reproductive toxicities. Increasingly research elucidated that BPA exposure to the environment would change the epigenetic modifications of transcriptome, but the mechanism by which BPA affects m6A methylation in interfering with female reproductive health remains uncertain. Therefore, this study preliminarily proposed and tested the hypothesis that BPA exposure alters the m6A modification level in transcripts in female ovarian granulosa cells. After BPA was exposed to granulosa cells for 24 h, RNA methylation related regulatory genes (such as METTL3, METTL14, ALKBH5, FTO) and the global m6A levels showed significant differences. Next, we applied MERIP-seq analysis to obtain information on the genome-wide m6A modification changes and identified 1595 differentially methylated mRNA transcripts, and 50 differentially methylated lncRNA transcripts. Further joint analysis of gene common expression showed that 33 genes were hypermethylated and up-regulated, 71 were hypermethylated and down-regulated, 49 were hypomethylated and up-regulated, and 20 were hypomethylated and down-regulated. Enriched Gene Ontology (GO) and biological pathway analysis revealed that these unique genes were mainly enriched in lipid metabolism, cell proliferation, and apoptosis related pathways. Six of these genes (mRNAs IMPA1, MCOLN1, DCTN3, BRCA2, and lncRNAs MALAT1, XIST) were validated using RT-qPCR and IGV software. Through comprehensive analysis of epitranscriptome and protein-protein interaction (PPI) data, lncRNAs MALAT1 and XIST are expected to serve as new markers for BPA interfering with the female reproductive system. In brief, these data show a novel and necessary connection between the damage of BPA exposure on female ovarian granulosa cells and RNA methylation modification.

N6-methyladenosine modification changes during the recovery processes for Paulownia witches' broom disease under the methyl methanesulfonate treatment.

Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N 6-methyladenosine (m6A) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the m6A transcriptome map of Paulownia fortunei (P. fortunei) has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on m6A modification of P. fortunei and obtained the whole transcriptome m6A map in P. fortunei by m6A-seq. The m6A-seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of m6A modification of P. fortunei. The correlation analysis between the RNA-seq and m6A-seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB-related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F-box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB-infected seedling treated with methyl methanesulfonate, and m6A modification was found in m6A-seq results. Moreover, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) verified that the alternative splicing of these two genes was associated with m6A modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.

Analysis of N6-methyladenosine reveals a new important mechanism regulating the salt tolerance of sugar beet (Beta vulgaris).

Salinity is an important environmental factor in crop growth and development. N6-methyladenosine (m6A) is an essential epigenetic modification that regulates plant-environment interaction. Sugar beet is a major sugar-yielding crop that has a certain tolerance to salt, but the dynamic response elicited by the m6A modification of transcripts under salt stress remains unknown. In this study, sugar beet was exposed to 300 mM NaCl to investigate its physiological response to high salinity and transcriptome-wide m6A modification profile. After the salt treatment, 7737 significantly modified m6A sites and 4981 differentially expressed genes (DEGs) were identified. Among the 312 m6A-modified DEGs, 113 hypomethylated DEGs were up-regulated and 99 hypermethylated DEGs were down-regulated, indicating a negative correlation between m6A modification and gene expression. Well-known salt tolerance genes (e.g., sodium/hydrogen exchanger 1, choline monooxygenase, and nucleoredoxin 2) and phospholipid signaling pathway genes (phosphoinositol-specific phospholipase C, phospholipase D, diacylglycerol kinase 1, etc.) were also among the m6A-modified genes. Further analysis showed that m6A modification may regulate salt-tolerant related gene expression by controlling mRNA stability. Therefore, changes in m6A modification may negatively regulate the expression of the salt-resistant genes in sugar beet, at least in part by modulating the stability of the mRNA via demethylase BvAlkbh10B. These findings could provide a better understanding of the epigenetic mechanisms of salt tolerance in sugar beets and uncover new candidate genes for improving the production of sugar beets planted in high-salinity soil.

The Important Role of m6A-Modified circRNAs in the Differentiation of Intramuscular Adipocytes in Goats Based on MeRIP Sequencing Analysis.

Intramuscular fat contributes to the improvement of goat meat quality. N6-Methyladenosine (m6A)-modified circular RNAs play important roles in adipocyte differentiation and metabolism. However, the mechanisms by which m6A modifies circRNA before and after differentiation of goat intramuscular adipocytes remain poorly understood. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and circRNA sequencing (circRNA-seq) to determine the distinctions in m6A-methylated circRNAs during goat adipocyte differentiation. The profile of m6A-circRNA showed a total of 427 m6A peaks within 403 circRNAs in the intramuscular preadipocytes group, and 428 peaks within 401 circRNAs in the mature adipocytes group. Compared with the intramuscular preadipocytes group, 75 peaks within 75 circRNAs were significantly different in the mature adipocytes group. Furthermore, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of intramuscular preadipocytes and mature adipocytes showed that the differentially m6A-modified circRNAs were enriched in the PKG signaling pathway, endocrine and other factor-regulated calcium reabsorption, lysine degradation, etc. m6A-circRNA-miRNA-mRNA interaction networks predicted the potential m6A-circRNA regulation mechanism in different goat adipocytes. Our results indicate that there is a complicated regulatory relationship between the 12 upregulated and 7 downregulated m6A-circRNAs through 14 and 11 miRNA mediated pathways, respectively. In addition, co-analysis revealed a positive association between m6A abundance and levels of circRNA expression, such as expression levels of circRNA_0873 and circRNA_1161, which showed that m6A may play a vital role in modulating circRNA expression during goat adipocyte differentiation. These results would provide novel information for elucidating the biological functions and regulatory characteristics of m6A-circRNAs in intramuscular adipocyte differentiation and could be helpful for further molecular breeding to improve meat quality in goats.

Transcriptome-wide high-throughput m6 A sequencing of differential m6 A methylation patterns in the decidual tissues from RSA patients.

Recurrent spontaneous abortion (RSA) is characterized by two or more consecutive pregnancy losses in the first trimester of pregnancy, experienced by 5% of women during their reproductive age. As a complex pathological process, the etiology of RSA remains poorly understood. Recent studies have established that gene expression changes dramatically in human endometrial stromal cells (ESCs) during decidualization. N6-methyladenosine (m6 A) modification is the most prevalent epigenetic modification of mRNA in eukaryotic cells and it is closely related to the occurrence and development of many pathophysiological phenomena. In this study, we first confirmed that high levels of m6 A mRNA methylation in decidual tissues are associated with RSA. Then, we used m6 A-modified RNA immunoprecipitation sequence (m6 A-seq) and RNA sequence (RNA-seq) to identify the differentially expressed m6 A methylation in decidual tissues from RSA patients and identified the key genes involved in abnormal decidualization by bioinformatics analysis. Using m6 A-seq, we identified a total of 2169 genes with differentially expressed m6 A methylation, of which 735 m6 A hypermethylated genes and 1434 m6 A hypomethylated genes were identified. Further joint analysis of m6 A-seq and RNA-seq revealed that 133 genes were m6 A modified with mRNA expression. GO and KEGG analyses indicated that these unique genes were mainly enriched in environmental information processing pathways, including the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathway. In summary, this study uncovered the transcriptome-wide m6 A modification pattern in decidual tissue of RSA, which provides a theoretical basis for further research into m6 A modification and new therapeutic strategies for RSA.

Analysis of m6A methylation in skin tissues of different sex Liaoning cashmere goats.

N6-methyladenosine (m6A) is the most frequent internal modification of mRNA and lncRNA in eukaryotes. We used two high-throughput sequencing method, m6A-seq and RNA-seq to identify pivotal m6A-modified genes in cashmere fineness and fiber growth. 8062 m6A peaks were detected by m6A-seq, including 2157 upregulated and 6445 downregulated. Furthermore, by comparing m6A-modified genes of the male Liaoning Cashmere Goat (M-LCG) and female Liaoning Cashmere Goat (F-LCG) skin tissues, we get 862 differentially expressed m6A-modified genes. To identify differently expressed m6A genes associated with cashmere fineness, 11 genes were selected for validation using real time fluorescent quantitative PCR in M-LCG and F-LCG. This study provides an acadamic basis on the molecular regulation mechanism of m6A modification in cashmere growth process.

Transcriptome-wide N6-methyladenosine (m6A) methylation in soybean under Meloidogyne incognita infection.

N6-methyladenosine (m6A) is a reversible epigenetic modification of mRNA and other RNAs that plays a significant role in regulating gene expression and biological processes. However, m6A abundance, dynamics, and transcriptional regulatory mechanisms remain unexplored in the context of soybean resistance to Meloidogyne incognita. In this study, we performed a comparative analysis of transcriptome-wide m6A and metabolome profiles of soybean root tissues with and without M. incognita infection. Global m6A hypermethylation was widely induced in response to M. incognita infection and was enriched around the 3' end of coding sequences and in 3' UTR regions. There were 2069 significantly modified m6A sites, 594 differentially expressed genes, and 103 differentially accumulated metabolites between infected and uninfected roots, including coumestrol, psoralidin, and 2-hydroxyethylphosphonate. Among 101 m6A-modified DEGs, 34 genes were hypomethylated and upregulated, and 39 genes were hypermethylated and downregulated, indicating a highly negative correlation between m6A methylation and gene transcript abundance. A number of these m6A-modified DEGs, including WRKY70, ERF60, POD47 and LRR receptor-like serine/threonine-protein kinases, were involved in plant defense responses. Our study provides new insights into the critical role of m6A modification in early soybean responses to M. incognita. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00077-2.

Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in the rat adenohypophysis after GnRH treatment.

N6-methyladenosine is considered to be the most common and abundant internal chemical modification among the more than 150 identified chemical RNA modifications. It is involved in most biological processes and actively participates in the regulation of animal reproduction. However, the potential function of m6 A in the pituitaries of mammals is not yet clear. It is also unknown whether m6 A is involved in the secretion and regulation of FSH by GnRH, which in turn affects mammalian reproduction. In this study, rats were treated with gonadorelin to simulate physiological GnRH-mediated regulation of FSH synthesis and secretion, and m6 A-seq was used to analyze the differential m6 A modification of the rat pituitary after gonadorelin treatment. A whole-transcriptome map of m6 A in the rat pituitary gland before and after gonadorelin treatment was successfully created. A total of 6413 differential peaks were identified, of which 3764 m6 A peaks were upregulated and 2649 m6 A peaks were downregulated. Among the 709 differentially expressed genes, 250 genes were discovered with differential methylation modifications. Intriguingly, the altered m6 A peaks within mRNAs were enriched in steroid biosynthetic processes and responses to cAMP. The results of the study will lay a foundation for further exploration of the potential role of m6 A modification in the regulation of reproductive hormone secretion and provide a theoretical basis for the application of GnRH analogs in mammalian artificial reproduction.

Alteration of N6-methyladenosine epitranscriptome profile in lipopolysaccharide-induced mouse mesangial cells.

N6-Methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in eukaryotes. The underlying molecular mechanisms of m6A modification in chronic glomerulonephritis (CGN) remain unexplored. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) analyses to assess the alterations of epitranscriptome-wide m6A profile in lipopolysaccharide (LPS)-induced mouse mesangial cells (MMC). The results of our data showed 2153 significantly differential m6A peaks and 358 significantly differentially expressed genes. Furthermore, integrated analysis from MeRIP-seq and RNA-seq identified a total of 64 genes with differential m6A modification and expressed levels, of which 5 genes displayed hypermethylation and upregulation, 42 genes displayed hypermethylation and downregulation, 11 genes displayed hypomethylation and upregulation, and 8 genes displayed hypomethylation and downregulation. Many of them (including Fosl1, Sorbs1, Ambp, Fgfr3, Nedd9, Fgg, Trim13, Fgf22, Mylk, and Muc6) are implicated in the regulation of the immune and inflammatory response. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis found that differential 64 genes were mainly enriched in fatty acid oxidation, apoptosis signaling pathway, complement and coagulation cascades, and PPAR signaling pathway. Together, our study provided a new perspective on the understanding of molecular features of m6A modification in CGN pathogenic pathogenesis.

m6A RNA Immunoprecipitation Followed by High-Throughput Sequencing to Map N6-Methyladenosine.

N6-methyladenosine (m6A) is the most abundant internal modification on messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) in eukaryotes. It influences gene expression by regulating RNA processing, nuclear export, mRNA decay, and translation. Hence, m6A controls fundamental cellular processes, and dysregulated deposition of m6A has been acknowledged to play a role in a broad range of human diseases, including cancer. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq or m6A-seq) is a powerful technique to map m6A in a transcriptome-wide level. After immunoprecipitation of fragmented polyadenylated (poly(A)+) rich RNA by using specific anti-m6A antibodies, both the immunoprecipitated RNA fragments together with the input control are subjected to massively parallel sequencing. The generation of such comprehensive methylation profiles of signal enrichment relative to input control is necessary in order to better comprehend the pathogenesis behind aberrant m6A deposition.

Transcriptome-wide N 6-Methyladenosine Methylome Profiling Reveals m6A Regulation of Skeletal Myoblast Differentiation in Cattle (Bos taurus).

N 6 -methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m6A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m6A-seq and RNA-seq to explore the m6A methylation modification pattern during bovine skeletal myogenesis. m6A-seq analysis revealed that m6A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691-8,094 m6A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m6A and mRNA levels, suggesting that m6A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m6A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m6A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m6A regulating skeletal muscle development.

Comprehensive Analysis of the Transcriptome-Wide m6A Methylation Modification Difference in Liver Fibrosis Mice by High-Throughput m6A Sequencing.

N6-Methyladenosine (m6A), a unique and common mRNA modification method in eukaryotes, is involved in the occurrence and development of many diseases. Liver fibrosis (LF) is a common response to chronic liver injury and may lead to cirrhosis and even liver cancer. However, the involvement of m6A methylation in the development of LF is still unknown. In this study, we performed a systematic evaluation of hepatic genome-wide m6A modification and mRNA expression by m6A-seq and RNA-seq using LF mice. There were 3,315 genes with significant differential m6A levels, of which 2,498 were hypermethylated and 817 hypomethylated. GO and KEGG analyses illustrated that differentially expressed m6A genes were closely correlated with processes such as the endoplasmic reticulum stress response, PPAR signaling pathway and TGF-ß signaling pathway. Moreover, a total of 90 genes had both a significant change in the m6A level and mRNA expression shown by joint analysis of m6A-seq and RNA-seq. Hence, the critical elements of m6A modification, including methyltransferase WTAP, demethylases ALKBH5 and binding proteins YTHDF1 were confirmed by RT-qPCR and Western blot. In an additional cell experiment, we also observed that the decreased expression of WTAP induced the development of LF as a result of promoting hepatic stellate cell (HSC) activation. Therefore, this study revealed unique differential m6A methylation patterns in LF mice and suggested that m6A methylation was associated with the occurrence and course of LF to some extent.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Genome-wide identification and characterization of circular RNA m6A modification in pancreatic cancer.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant modification of RNA in eukaryotic cells and play critical roles in cancer. While most related studies focus on m6A modifications in linear RNA, transcriptome-wide profiling and exploration of m6A modification in circular RNAs in cancer is still lacking. METHODS: For the detection of m6A modification in circRNAs, we developed a new bioinformatics tools called Circm6A and applied it to the m6A-seq data of 77 tissue samples from 58 individuals with pancreatic ductal adenocarcinoma (PDAC). RESULTS: Circm6A performs better than the existing circRNA identification tools, which achieved highest F1 score among these tools in the detection of circRNAs with m6A modifications. By using Circm6A, we identified a total of 8807 m6A-circRNAs from our m6A-seq data. The m6A-circRNAs tend to be hypermethylated in PDAC tumor tissues compared with normal tissues. The hypermethylated m6A-circRNAs were associated with a significant gain of circRNA-mRNA coexpression network, leading to the dysregulation of many important cancer-related pathways. Moreover, we found the cues that hypermethylated m6A-circRNAs may promote the circularization and translation of circRNAs. CONCLUSIONS: These comprehensive findings further bridged the knowledge gaps between m6A modification and circRNAs fields by depicting the m6A-circRNAs genomic landscape of PDAC patients and revealed the emerging roles played by m6A-circRNAs in pancreatic cancer. Circm6A is available at https://github.com/canceromics/circm6a .

Transcriptome-wide N6-methyladenosine (m6A) methylation in watermelon under CGMMV infection.

BACKGROUND: Cucumber green mottle mosaic virus (CGMMV) causes substantial global losses in cucurbit crops, especially watermelon. N6-methyladenosine (m6A) methylation in RNA is one of the most important post-transcriptional modification mechanisms in eukaryotes. It has been shown to have important regulatory functions in some model plants, but there has been no research regarding m6A modifications in watermelon. RESULTS: We measured the global m6A level in resistant watermelon after CGMMV infection using a colorimetric method. And the results found that the global m6A level significantly decreased in resistant watermelon after CGMMV infection. Specifically, m6A libraries were constructed for the resistant watermelon leaves collected 48 h after CGMMV infection and the whole-genome m6A-seq were carried out. Numerous m6A modified peaks were identified from CGMMV-infected and control (uninfected) samples. The modification distributions and motifs of these m6A peaks were highly conserved in watermelon transcripts but the modification was more abundant than in other reported crop plants. In early response to CGMMV infection, 422 differentially methylated genes (DMGs) were identified, most of which were hypomethylated, and probably associated with the increased expression of watermelon m6A demethylase gene ClALKBH4B. Gene Ontology (GO) analysis indicated quite a few DMGs were involved in RNA biology and stress responsive pathways. Combined with RNA-seq analysis, there was generally a negative correlation between m6A RNA methylation and transcript level in the watermelon transcriptome. Both the m6A methylation and transcript levels of 59 modified genes significantly changed in response to CGMMV infection and some were involved in plant immunity. CONCLUSIONS: Our study represents the first comprehensive characterization of m6A patterns in the watermelon transcriptome and helps to clarify the roles and regulatory mechanisms of m6A modification in watermelon in early responses to CGMMV.

Transcriptome-Wide N6-Methyladenosine (m6A) Profiling of Susceptible and Resistant Wheat Varieties Reveals the Involvement of Variety-Specific m6A Modification Involved in Virus-Host Interaction Pathways.

N6-methyladenosine (m6A) methylation is the most prevalent internal modification of post-transcriptional modifications in mRNA, tRNA, miRNA, and long non-coding RNA in eukaryotes. m6A methylation has been proven to be involved in plant resistance to pathogens. However, there are no reports on wheat (Triticum aestivum) m6A transcriptome-wide map and its potential biological function in wheat resistance to wheat yellow mosaic virus (WYMV). To the best of our knowledge, this study is the first to determine the transcriptome-wide m6A profile of two wheat varieties with different resistances to WYMV. By analyzing m6A-sequencing (m6A-seq) data, we identified 25,752 common m6A peaks and 30,582 common m6A genes in two groups [WYMV-infected resistant wheat variety (WRV) and WYMV-infected sensitive wheat variety (WSV)], and all these peaks were mainly enriched in 3' untranslated regions and stop codons of coding sequences. Gene Ontology analysis of m6A-seq and RNA-sequencing data revealed that genes that showed significant changes in both m6A and mRNA levels were associated with plant defense responses. Kyoto Encyclopedia of Genes and Genomes analysis revealed that these selected genes were enriched in the plant-pathogen interaction pathway. We further verified these changes in m6A and mRNA levels through gene-specific m6A real-time quantitative PCR (RT-qPCR) and normal RT-qPCR. This study highlights the role of m6A methylation in wheat resistance to WYMV, providing a solid basis for the potential functional role of m6A RNA methylation in wheat resistance to infection by RNA viruses.

Comprehensive Analysis of Differential m6A RNA Methylomes in the Hippocampus of Cocaine-Conditioned Mice.

N6-methyladenosine (m6A) is the most prevalent internal modification found in mRNAs and lncRNA and plays a vital role in posttranscriptional regulation in mammals. m6A is abundant in the nervous system, where it modulates neuronal development and hippocampus-dependent learning and memory. However, the roles of RNAs m6A modification and its related enzymes in cocaine reward are still not fully understood. In this study, we found that the fat mass and obesity-associated gene (FTO) demethylase, but not methyltransferase-like 3 (METTL3) and 14 (METTL14), was downregulated in the hippocampus following cocaine-induced conditioned place preference (CPP), and the level of m6A is notably higher in the hippocampus of cocaine CPP training mice. Using methylated m6A RNA immunoprecipitation sequencing (MeRIP-m6A-seq), we identified a total of 6516 m6A peaks within 4460 mRNAs, and 3083 m6A peaks within 850 lncRNAs were significantly dysregulated. Intriguingly, the altered m6A peaks within mRNAs and lncRNAs were enriched in synapse maturation and localization processes. Our study uncovers a critical role for an m6A epitranscriptomic dysregulation and downregulation of FTO expression in the hippocampus following cocaine-induced CPP.

Transcriptome-Wide High-Throughput m6A Sequencing of Differential m6A Methylation Patterns in the Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes Cell Line MH7A.

INTRODUCTION: N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic mRNAs and is closely related to the occurrence and development of many diseases, especially tumors. However, the relationship between m6A methylation and rheumatoid arthritis (RA) is still a mystery. METHODS: Two high-throughput sequencing methods, namely, m6A modified RNA immunoprecipitation sequence (m6A-seq) and RNA sequence (RNA-seq) were performed to identify the differentially expressed m6A methylation in human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A after stimulation with TNF-α. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to obtain enriched GO terms and significant KEGG pathways. Then, four candidate genes, Wilms tumor 1-associating protein (WTAP), receptor-interacting serine/threonine protein kinase 2 (RIPK2), Janus kinase 3 (JAK3) and tumor necrosis factor receptor SF10A (TNFRSF10A) were selected to further validate the m6A methylation, mRNA and protein expression levels in MH7A cells and synovial tissues of adjuvant arthritis (AA) rats by RT-qPCR and Western blot. RESULTS: Using m6A-seq, we identified a total of 206 genes with differentially expressed m6A methylation, of which 118 were significantly upregulated and 88 genes were significantly downregulated. Likewise, 1207 differentially mRNA expressed mRNAs were obtained by RNA-seq, of which 793 were upregulated and 414 downregulated. Further joint analysis showed that the m6A methylation and mRNA expression levels of 88 genes changed significantly, of which 30 genes displayed increased m6A methylation and decreased mRNA expression, 57 genes displayed decreased m6A methylation and increased mRNA expression increased, and 1 gene displayed increased m6A methylation and increased mRNA expression. GO and KEGG analyses indicated that these unique genes were mainly enriched in inflammation-related pathways, cell proliferation and apoptosis. In addition, the validations of WTAP, RIPK2, JAK3 and TNFRSF10A were in accordance with the m6A and RNA sequencing results. CONCLUSION: This study established the transcriptional map of m6A in MH7A cells and revealed the potential relationship between RNA methylation modification and RA related genes. The results suggested that m6A modification was associated with the occurrence and course of RA to some extent.