Transcriptome-wide Mapping of Internal N7-Methylguanosine Methylome in Mammalian mRNA.

N7-methylguanosine (m7G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.

Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N7-methylguanosine (m7G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m7G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m7G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m7G tRNA methylome and highlights its essential role in ESCs with links to human disease.

Evaluation of epitranscriptome-wide N6-methyladenosine differential analysis methods.

RNA methylation has emerged recently as an active research domain to study post-transcriptional alteration in gene expression regulation. Various types of RNA methylation, including N6-methyladenosine (m6A), are involved in human disease development. As a newly developed sequencing biotechnology to quantify the m6A level on a transcriptome-wide scale, MeRIP-seq expands RNA epigenetics study in both basic and clinical applications, with an upward trend. One of the fundamental questions in RNA methylation data analysis is to identify the Differentially Methylated Regions (DMRs), by contrasting cases and controls. Multiple statistical approaches have been recently developed for DMR detection, but there is a lack of a comprehensive evaluation for these analytical methods. Here, we thoroughly assess all eight existing methods for DMR calling, using both synthetic and real data. Our simulation adopts a Gamma-Poisson model and logit linear framework, and accommodates various sample sizes and DMR proportions for benchmarking. For all methods, low sensitivities are observed among regions with low input levels, but they can be drastically boosted by an increase in sample size. TRESS and exomePeak2 perform the best using metrics of detection precision, FDR, type I error control and runtime, though hampered by low sensitivity. DRME and exomePeak obtain high sensitivities, at the expense of inflated FDR and type I error. Analyses on three real datasets suggest differential preference on identified DMR length and uniquely discovered regions, between these methods.

ALKBH5 regulates chicken adipogenesis by mediating LCAT mRNA stability depending on m6A modification.

BACKGROUND: Previous studies have demonstrated the role of N6-methyladenosine (m6A) RNA methylation in various biological processes, our research is the first to elucidate its specific impact on LCAT mRNA stability and adipogenesis in poultry. RESULTS: The 6 100-day-old female chickens were categorized into high (n = 3) and low-fat chickens (n = 3) based on their abdominal fat ratios, and their abdominal fat tissues were processed for MeRIP-seq and RNA-seq. An integrated analysis of MeRIP-seq and RNA-seq omics data revealed 16 differentially expressed genes associated with to differential m6A modifications. Among them, ELOVL fatty acid elongase 2 (ELOVL2), pyruvate dehydrogenase kinase 4 (PDK4), fatty acid binding protein 9 (PMP2), fatty acid binding protein 1 (FABP1), lysosomal associated membrane protein 3 (LAMP3), lecithin-cholesterol acyltransferase (LCAT) and solute carrier family 2 member 1 (SLC2A1) have ever been reported to be associated with adipogenesis. Interestingly, LCAT was down-regulated and expressed along with decreased levels of mRNA methylation methylation in the low-fat group. Mechanistically, the highly expressed ALKBH5 gene regulates LCAT RNA demethylation and affects LCAT mRNA stability. In addition, LCAT inhibits preadipocyte proliferation and promotes preadipocyte differentiation, and plays a key role in adipogenesis. CONCLUSIONS: In conclusion, ALKBH5 mediates RNA stability of LCAT through demethylation and affects chicken adipogenesis. This study provides a theoretical basis for further understanding of RNA methylation regulation in chicken adipogenesis.

High N6-methyladenosine-activated TCEAL8 mRNA is a novel pancreatic cancer marker.

N6-methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells, m6A is upregulated, contributing to their malignant transformation. In this study, we analyzed gene expression and m6A modification in cancer tissues, ducts, and acinar cells derived from pancreatic cancer patients using MeRIP-seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them, the m6A-activated mRNA TCEAL8 was observed, for the first time, as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells, and activation of cancer-related signaling pathways was observed relative to TCEAL8-negative cells. Furthermore, among TCEAL8-positive cells, the cells expressing the m6A-modifying enzyme gene METTL3 showed co-activation of Notch and mTOR signaling, also known to be involved in cancer metastasis. Overall, these results suggest that m6A-activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer.

Comprehensive analysis of m6A modification in lipopolysaccharide-induced acute lung injury in mice.

BACKGROUND: N6-Methyladenosine (m6A) methylation is the most prevalent post-transcriptional modification in mRNA, and plays significant roles in various diseases. Nevertheless, the precise functions of m6A modification in the formation of ALI remain unclear. In this study we explore the transcriptome distribution of m6A methylation and its probable roles of in ALI. METHODS: Lipopolysaccharide (LPS) was utilized to establish an ALI mouse model. Real-time qPCR, Western blotting and m6A dot blot were utilized to assess m6A methylation level and the expression of m6A methylation enzymes. MeRIP-Seq and RNA-seq were utilized to explore differential m6A modifications and differentially expressed genes in ALI mice. The hub genes and enriched pathways were assessed by Real-time qPCR and Western blotting. RESULTS: Our findings showed that overall m6A methylation level was increased in ALI mice lung tissues, accompanied by lower levels of METTL3 and FTO. Notably, the protein expression of these methylases were different in various cells. There were 772 differently expressed m6A peaks in ALI as compared to the control group, with 316 being hypermethylated and 456 being hypomethylated. GO and KEGG analyses demonstrated these differentially methylated genes were associated with the calcium signaling pathway and cAMP signaling pathway. Furthermore, we identified 50 genes with distinct m6A peaks and mRNA expressions by combined analysis of MeRIP-Seq and RNA-Seq. KEGG analysis also demonstrated that these overlapped genes were closely associated with the calcium signaling pathway, cGMP-PKG signaling pathway, etc. Besides, Western blotting results demonstrated that the protein expression of Fibronectin leucine-rich transmembrane protein 3 (Flrt3) as well as the calcium signaling pathway and cGMP-PKG signaling pathway, increased significantly after ALI. CONCLUSIONS: m6A modification was paramount in the pathogenesis of ALI, and provided a foundation for the further investigation in the prevention and treatment of ALI.

N6-methyladenosine modification positively regulate Japanese encephalitis virus replication.

N6-methyladenosine (m6A) is present in diverse viral RNA and plays important regulatory roles in virus replication and host antiviral innate immunity. However, the role of m6A in regulating JEV replication has not been investigated. Here, we show that the JEV genome contains m6A modification upon infection of mouse neuroblast cells (neuro2a). JEV infection results in a decrease in the expression of m6A writer METTL3 in mouse brain tissue. METTL3 knockdown by siRNA leads to a substantial decrease in JEV replication and the production of progeny viruses at 48 hpi. Mechanically, JEV triggered a considerable increase in the innate immune response of METTL3 knockdown neuro2a cells compared to the control cells. Our study has revealed the distinctive m6A signatures of both the virus and host in neuro2a cells infected with JEV, illustrating the positive role of m6A modification in JEV infection. Our study further enhances understanding of the role of m6A modification in Flaviviridae viruses.

Transcriptomic evaluation of N6-methyladenosine modification can be used to identify differentially gene and immune-related biological processes in TX mice with liver fibrosis.

BACKGROUND: N6-methyladenosine (m6A) modification controls the stability, splicing, and translation of mRNA, which is important in the development of illnesses. Wilson's disease (WD) is an autosomal recessive liver copper metabolic disorder that causes liver fibrosis. The role of m6A methylation in WD-induced liver fibrosis development is still unclear. Thus, the goal of this study was to examine the scope of m6A methylation and further explore the potential targets related to WD-induced liver fibrosis. RESULTS: A total of 1930 significantly different m6A peaks were found on 1737 mRNAs, of which 993 were hypermethylated and 744 were hypomethylated when comparing normal and WD-induced liver fibrosis mice (n = 3). In parallel, 1261 differentially expressed mRNAs, comprising 557 upregulated and 704 downregulated mRNAs, were found. Overall, 114 mRNAs with significant changes in m6A levels and RNA expression were identified via joint analysis. Then, through PPI network construction and functional enrichment analysis, 12 hub genes were identified, these genes were mainly enriched in the inflammatory response and immunomodulation, and they are associated with immune cell infiltration. CONCLUSIONS: The significant difference in the amount of mRNA m6A modifications indicates that m6A modification is involved in the progression of WD-induced liver fibrosis, and theidentified hub genes are involved in inflammation and immune infiltration. These results may provide insights for subsequent studies on potential regulatory mechanisms.

N6-Methyladenosine (m6A) Sequencing Reveals Heterodera glycines-Induced Dynamic Methylation Promoting Soybean Defense.

Unraveling the intricacies of soybean cyst nematode (Heterodera glycines) race 4 resistance and susceptibility in soybean breeding lines-11-452 (highly resistant) and Dongsheng1 (DS1, highly susceptible)-was the focal point of this study. Employing cutting-edge N6-methyladenosine (m6A) and RNA sequencing techniques, we delved into the impact of m6A modification on gene expression and plant defense responses. Through the evaluation of nematode development in both resistant and susceptible roots, a pivotal time point (3 days postinoculation) for m6A methylation sequencing was identified. Our sequencing data exhibited robust statistics, successful soybean genome mapping, and prevalent m6A peak distributions, primarily in the 3' untranslated region and stop codon regions. Analysis of differential methylation peaks and differentially expressed genes revealed distinctive patterns between resistant and susceptible genotypes. In the highly resistant line (11-452), key resistance and defense-associated genes displayed increased expression coupled with inhibited methylation, encompassing crucial players such as R genes, receptor kinases, and transcription factors. Conversely, the highly susceptible DS1 line exhibited heightened expression correlated with decreased methylation in genes linked to susceptibility pathways, including Mildew Locus O-like proteins and regulatory elements affecting defense mechanisms. Genome-wide assessments, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and differential methylation peak/differentially expressed gene overlap emphasized the intricate interplay of m6A modifications, alternative splicing, microRNA, and gene regulation in plant defense. Protein-protein interaction networks illuminated defense-pivotal genes, delineating divergent mechanisms in resistant and susceptible responses. This study sheds light on the dynamic correlation between methylation, splicing, and gene expression, providing profound insights into plant responses to nematode infection.

Comprehensive analysis of transcriptome-wide N6-methyladenosine methylomes in the Barrett's esophagus in rats.

PURPOSE: As the most abundant RNA modification, N6-methyladenosine (m6A) methylation plays crucial roles in various diseases. The aim of this study is to comprehensively map the landscape of the mRNA m6A modification pattern in Barrett's esophagus (BE) in order to find key genes and potential therapy for BE and even esophageal adenocarcinoma (EAC). METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were performed to compare the difference in mRNA m6A methylation and differentially expressed mRNAs between BE and normal control (NC) tissues. Bioinformatics analysis was used to describe the m6A modification pattern and specific genes in BE and NC tissues. RESULTS: Through MeRIP-seq, we obtained m6A methylation profiling in BE and NC tissues. In total, 11,026 unique peaks were detected in the BE groups, whereas 8564 unique peaks were detected in the NC groups. Peaks were primarily enriched within CDS with GGACU motifs and most of the peaks were within 1000 bp in width. Moreover, functional enrichment analysis demonstrated that hypermethylated and hypomethylated genes were significantly enriched in coronavirus disease pathway, calcium signaling pathway and MAPK signaling pathways. Furthermore, PPI network was conducted and 18 hub genes were identified via STRING database and Cystoscope. Among them, ACTA1, CDC20, CKM, KIF20a, MYH11, TPM2, MYL9, DES, TNNT3 were overexpressed in EAC in the GEPIA gene bank and TPM1, KIF20a impaired patients' survival in the Kaplan-Meier plotter database. Finally, functional enrichment analysis demonstrated that co-expressed genes of TPM1 were significantly enriched in calcium signaling pathway, cGMP-PKG signaling pathway and PI3K-Akt signaling pathway. CONCLUSION: Our study is the first to perform comprehensive and transcriptome-wide maps to identify the potential roles played by m6A methylation in BE, which widely involved in oxidative stress. This foresees a guiding role in revealing the molecular mechanism of m6A-mediated genes that govern the pathogenesis and progression of BE and EAC.

Dynamic alterations of the transcriptome-wide m6A methylome in cytogenetically normal acute myeloid leukaemia during initial diagnosis and relapse.

Accumulating studies have indicated that N6-methyladenosine (m6A) plays an important role in acute myeloid leukaemia (AML). However, little is known about the m6A methylome at a transcriptome-wide scale in AML patients. We obtained three pairs of bone marrow (BM) samples from cytogenetically normal AML patients at the timepoints of diagnosis (AML) and relapse (R_AML) and three BM samples from healthy donors used as normal controls (NCs). Methylated RNA immunoprecipitation next-generation sequencing (MeRIP-Seq) was conducted to identify differences in the m6A methylomes between AML and NC and between R_AML and AML. We identified a total of 11,076 and 11,962 differential m6A peaks in AML and R_AML group, respectively. These dysregulated m6A peaks were detected on all chromosomes, especially chr1, chr19 and chr17, and were mainly enriched in 3' untranslated regions, stop codon and coding sequence regions. Moreover, GO and KEGG analyses indicated that m6A -modified genes were significantly enriched in cancer-related biological functions and pathways. Additionally, we identified a link between the m6A methylome and RNA transcriptome via combined analyses of MeRIP-seq and RNA-seq data. In addition, 5 genes, HSPG2, HOMER3, TSPO2, CXCL12 and FUT1 regulated by m6A modification potentially, were shown to be related to the prognosis of AML patients. Additionally, we detected the mRNA expression of major m6A regulators and potential target mRNA on the leukemogenesis and found that the expression of IGF2BP2, HSPG2 and HOMER3 were upregulated in AML at the time of diagnosis. Moreover, their expression became downregulated after remission and then elevated again at relapse. Our study provides the first data on the differential m6A methylome in AML patients during initial diagnosis and relapse. This study demonstrates a novel relationship between m6A modification and AML relapse and paves the way for further studies aimed at elucidating the epigenic mechanisms involved in the relapse of AML.

Transcriptome-wide N6-methyladenosine methylation profile of atherosclerosis in mice.

BACKGROUND: Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N6-methyladenosine (m6A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m6A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m6A modification in AS and its potential mechanism. METHODS: Methylation Quantification Kit was used to detect the global m6A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m6A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m6A levels in target genes. RESULTS: The global m6A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m6A methylation maps in AS and control mice. In total, 26,918 m6A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m6A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m6A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m6A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m6A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene Fabp5 may be a potential biomarker of AS. CONCLUSIONS: Our study provides transcriptome-wide m6A methylation for the first time to determine the association between m6A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.

Identification and analysis of RNA-5-methylcytosine-related key genes in osteoarthritis.

BACKGROUND: 5-methylcytosine (m5C) modification is widely associated with many biological and pathological processes. However, knowledge of m5C modification in osteoarthritis (OA) remains lacking. Thus, our study aimed to identify common m5C features in OA. RESULTS: In the present study, we identified 1395 differentially methylated genes (DMGs) and 1673 differentially expressed genes (DEGs) using methylated RNA immunoprecipitation next-generation sequencing (MeRIP-seq) and RNA-sequencing. A co-expression analysis of DMGs and DEGs showed that the expression of 133 genes was significantly affected by m5C methylation. A protein-protein interaction network of the 133 genes was constructed using the STRING database, and the cytoHubba plug-in of Cytoscape was used to hub genes were screen out 11 hub genes, including MMP14, VTN, COL15A1, COL6A2, SPARC, COL5A1, COL6A3, COL6A1, COL8A2, ADAMTS2 and COL7A1. The Pathway enrichment analysis by the ClueGO and CluePedia plugins in Cytoscape showed that the hub genes were significantly enriched in collagen degradation and extracellular matrix degradation. CONCLUSIONS: Our study indicated that m5C modification might play an important role in OA pathogenesis, and the present study provides worthwhile insight into identifying m5C-related therapeutic targets in OA.

Comprehensive analysis of N6-methyladenosine-related RNA methylation in the mouse hippocampus after acquired hearing loss.

BACKGROUND: The mechanism underlying cognitive impairment after hearing loss (HL) remains unclear. N6-methyladenosine (m6A) is involved in many neurodegenerative diseases; however, its role in cognitive impairment after HL has not yet been investigated. Therefore, we aimed to analyze the m6A modification profile of the mouse hippocampus after HL exposure. A mouse model of neomycin-induced HL was established. An auditory brainstem-response test was utilized for detecting hearing threshold. The passive avoidance test was served as the mean for evaluating cognitive function. The m6A-regulated enzyme expression levels were analyzed by using reverse transcription quantitative real-time polymerase chain reaction and western blot analyses. RNA sequencing (RNA-Seq) and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were performed with the aim of investigating gene expression differences and m6A modification in the mouse hippocampus. RESULTS: Neomycin administration induced severe HL in mice. At four months of age, the mice in the HL group showed poorer cognitive performance than the mice in the control group. METTL14, WTAP, and YTHDF2 mRNA levels were downregulated in the hippocampi of HL mice, whereas ALKBH5 and FTO mRNA levels were significantly upregulated. At the protein level, METTL3 and FTO were significantly upregulated. Methylated RNA immunoprecipitation sequencing analysis revealed 387 and 361 m6A hypermethylation and hypomethylation peaks, respectively. Moreover, combined analysis of mRNA expression levels and m6A peaks revealed eight mRNAs with significantly changed expression levels and methylation. CONCLUSIONS: Our findings revealed the m6A transcriptome-wide profile in the hippocampus of HL mice, which may provide a basis for understanding the association between HL and cognitive impairment from the perspective of epigenetic modifications.

Transcriptome-wide analysis of RNA m6A methylation regulation of muscle development in Queshan Black pigs.

BACKGROUND: N6-methyladenosine (m6A) refers to the methylation modification of N6 position of RNA adenine, a dynamic reversible RNA epigenetic modification that plays an important regulatory role in a variety of life processes. In this study, we used MeRIP-Seq and RNA-Seq of the longissimus dorsi (LD) muscle of adult (QA) and newborn (QN) Queshan Black pigs to screen key genes with m6A modification involved in muscle growth by bioinformatics analysis. RESULTS: A total of 23,445 and 25,465 m6A peaks were found in the whole genomes of QA and QN, respectively. Among them, 613 methylation peaks were significantly different (DMPs) and 579 genes were defined as differentially methylated genes (DMGs). Compared with the QN group, there were 1,874 significantly differentially expressed genes (DEGs) in QA group, including 620 up-regulated and 1,254 down-regulated genes. In order to investigate the relationship between m6A and mRNA expression in the muscle of Queshan Black pigs at different periods, a combined analysis of MeRIP-Seq and RNA-Seq showed that 88 genes were significantly different at both levels. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes results showed that DEGs and DMGs were mainly involved in skeletal muscle tissue development, FoxO signaling pathway, MAPK signaling pathway, insulin signaling pathway, PI3K-Akt signaling pathway, and Wnt signaling pathway. Four DEGs (IGF1R, CCND2, MYOD1 and FOS) and four DMGs (CCND2, PHKB, BIN1 and FUT2), which are closely related to skeletal muscle development, were selected as candidate genes for verification, and the results were consistent with the sequencing results, which indicated the reliability of the sequencing results. CONCLUSIONS: These results lay the foundation for understanding the specific regulatory mechanisms of growth in Queshan Black pigs, and provide theoretical references for further research on the role of m6A in muscle development and breed optimization selection.

Transcriptome-wide 5-methylcytosine modification profiling of long non-coding RNAs in A549 cells infected with H1N1 influenza A virus.

BACKGROUND: In recent years, accumulating evidences have revealed that influenza A virus (IAV) infections induce significant differential expression of host long noncoding RNAs (lncRNAs), some of which play important roles in the regulation of virus-host interactions and determining the virus pathogenesis. However, whether these lncRNAs bear post-translational modifications and how their differential expression is regulated remain largely unknown. In this study, the transcriptome-wide 5-methylcytosine (m5C) modification of lncRNAs in A549 cells infected with an H1N1 influenza A virus was analyzed and compared with uninfected cells by Methylated RNA immunoprecipitation sequencing (MeRIP-Seq). RESULTS: Our data identified 1317 upregulated m5C peaks and 1667 downregulated peaks in the H1N1 infected group. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially modified lncRNAs were associated with protein modification, organelle localization, nuclear export and other biological processes. Furthermore, conjoint analysis of the differentially modified (DM) and differentially expressed (DE) lncRNAs identified 143 'hyper-up', 81 'hypo-up', 6 'hypo-down' and 4 'hyper-down' lncRNAs. GO and KEGG analyses revealed that these DM and DE lncRNAs were predominantly associated with pathogen recognition and disease pathogenesis pathways, indicating that m5C modifications could play an important role in the regulation of host response to IAV replication by modulating the expression and/or stability of lncRNAs. CONCLUSION: This study presented the first m5C modification profile of lncRNAs in A549 cells infected with IAV and demonstrated a significant alteration of m5C modifications on host lncRNAs upon IAV infection. These data could give a reference to future researches on the roles of m5C methylation in virus infection.

Altered 5-methylcytosine modification of mRNA is involved in the pathogenesis of pre-eclampsia.

5-Methylcytosine (m5 C) is a prevalent RNA modification in messenger RNAs (mRNAs). Despite its abundance, its role in the decidua of pre-eclampsia (PE) remains elusive. In this study, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) to map m5 C peaks and mRNA expression profile in the decidua of human early-onset PE (EPE), late-onset PE (LPE), and normal pregnancy (NP). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses elucidated potential roles of the differentially methylated mRNAs (DMGs) and differentially expressed mRNAs in decidualization pathways. Integrative analysis of MeRIP-seq and RNA-seq data pinpointed 50 candidate genes linked to PE, marked by both differentially methylated m5 C peaks and congruent expression changes. To validate these observations, we selected nine genes for verification via quantitative PCR. Our results underscore the precision and reproducibility of our bioinformatics approach. Importantly, we propose that changes in m5 C modification and expression of relevant mRNA might influence the pathogenesis of PE by hampering decidualization. This work shines light on the distinct mRNA m5 C modification patterns and expression profiles in the decidua of PE, implicating pivotal signaling disruptions and decidualization impediments in the onset of PE.

m6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

BACKGROUND: Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. METHODS AND RESULTS: In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25 °C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5 °C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. CONCLUSIONS: m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

Integrated analysis of transcriptome-wide m6A methylation in a Cd-induced kidney injury rat model.

BACKGROUND: Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in a variety of diseases. However, the specific functions of m6A in CdCl2-induced kidney injury remain unclear. OBJECTIVE: Here, we investigate a transcriptome-wide map of m6A modifications and explore the effects of m6A on Cd-induced kidney injury. MATERIALS AND METHODS: The rat kidney injury model was constructed by subcutaneous injection of CdCl2 (0.5, 1.0, and 2.0 mg/kg). The m6A levels were measured by colorimetry. The level of expression of m6A-related enzymes were detected by reverse transcription quantitative real-time PCR analysis. Transcriptome-wide m6A methylome in CdCl2 (2.0 mg/kg) and the control group were profiled by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Subsequently, the sequencing data were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), while gene set enrichment analysis (GSEA) confirmed the functional enrichment pathways of sequencing genes. In addition, a protein-protein interaction (PPI) network was applied to select hub genes. RESULTS: The levels of m6A and m6A regulators (METTL3, METTL14, WTAP, YTHDF2) were significantly increased in CdCl2 groups. We identified a total of 2615 differentially expressed m6A peaks, 868 differentially expressed genes and 200 genes with significant changes in both m6A modification and gene expression levels. GO, KEGG, and GSEA analyses indicated that these genes were mainly enriched in inflammation and metabolism-related pathways such as in IL-17 signaling and fatty acid metabolism. According the result of the conjoint analysis, we identified the top ten hub genes (Fos, Hsp90aa1, Gata3, Fcer1g, Cftr, Cspg4, Atf3, Cdkn1a, Ptgs2, and Npy) which may be regulated by m6A and involve in CdCl2-induced kidney damage. CONCLUSION: This study established a m6A transcriptional map in a CdCl2-induced kidney injury model and suggested that m6A may affect CdCl2 induced kidney injury via regulated the inflammation and metabolism related gene.

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.