Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination
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ABSTRACT:
ORGANISM(S): Mus musculus Escherichia coli synthetic construct Homo sapiens
PROVIDER: GSE201064 | GEO | 2022/10/24
REPOSITORIES: GEO
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Project description:We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome.
2022-10-24 | GSE201062 | GEO
Project description:We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome.
2022-10-24 | GSE201063 | GEO
Project description:We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome.
2022-10-24 | GSE211303 | GEO
Project description:Background: In higher eukaryotes, N6-methyladenosine (m6A) is the most common internal form of messenger RNA modification. By mapping the m6A methyl genome in multiple species, the potential regulatory function of reversible m6A methylation on mRNA is revealed. Recent studies have shown that RNA m6A modification influences mRNA transcription, location, translation, stability, splicing, and nuclear export. However, there are not enough data on the m6A transcriptome-wide map and its potential biological role in the heat stress of Pak-choi (Brassica rapa ssp. chinensis). Methods: In this work, MeRIP-seq was used to obtain the first transcriptome-wide profiling of RNA m6A modification in Pak-choi. Meanwhile, the transcriptome data were obtained by analyzing the input samples' sequencing data. Results: Our research indicated that with three replicates, there were 11,252 common m6A peaks and 9729 common m6A-containing genes identified in the normal (CK) and heat stress (T43) groups. It was found that m6A peaks were highly enriched in the 3' untranslated region in both CK and T43 groups. About 80% of the genes have one m6A site. The consensus sequence of m6A peaks was also enriched, which showed as AAACCV (V: U/A/G). In addition, association analysis found that there is a certain relationship between the degree of m6A methylation and the transcription level, indicating that m6A plays a certain regulatory role in gene expression. Conclusion: This comprehensive map in the study may provide a solid basis for determining the potential function of RNA m6A modification in Pak-choi under normal (CK) and heat stress (T43) conditions.
| S-EPMC7570095 | biostudies-literature
Project description:N6-methyladenosine (m6A) modification is the most extensively studied epigenetic modification due to its crucial role in regulating an array of biological processes. Herein, Bsu06560, formerly annotated as an adenine deaminase derived from Bacillus subtilis 168, was recognized as the first enzyme capable of metabolizing the epigenetic nucleoside N6-methyladenosine. A model of Bsu06560 was constructed, and several critical residues were putatively identified via mutational screening. Two mutants, F91L and Q150W, provided a superiorly enhanced conversion ratio of adenosine and N6-methyladenosine. The CRISPR-Cas9 system generated Bsu06560-knockout, F91L, and Q150W mutations from the B. subtilis 168 genome. Transcriptional profiling revealed a higher global gene expression level in BS-F91L and BS-Q150W strains with enhanced N6-methyladenosine deaminase activity. The differentially expressed genes were categorized using GO, COG, KEGG and verified through RT-qPCR. This study assessed the crucial roles of Bsu06560 in regulating adenosine and N6-methyladenosine metabolism, which influence a myriad of biological processes. This is the first systematic research to identify and functionally annotate an enzyme capable of metabolizing N6-methyladenosine and highlight its significant roles in regulation of bacterial metabolism. Besides, this study provides a novel method for controlling gene expression through the mutations of critical residues.
| S-EPMC8643624 | biostudies-literature
Project description:We applied chemical-assisted, m6A-SEAL method to profile m6A distributions in the transcriptomes of human and plant cells and compared its results against MeRIPm6A-seq.
2020-04-22 | GSE129979 | GEO
Project description:BackgroundThe newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek's disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells.ResultsMethylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection.ConclusionsIn this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.
| S-EPMC8063467 | biostudies-literature
Project description:In mammals, skeletal muscle development is a complex biological process regulated by many factors. N6-methyladenosine (m6A) RNA modification plays an important role in many biological processes. However, the regulation of m6A on skeletal muscle growth and development in adult goats remains unclear. In this study, Duan goats (DA) and Nubia goats (NBY), both female and 12 months old, were selected as the research objects, and m6A-Seq and RNA-Seq were mainly used to detect the difference of m6A modification and gene expression during the development of the longissimus dorsi (LD) muscle in the two breeds. The results showed that compared with DA, the meat production performance of NBY was better than that of DA, and the modification level of m6A was higher than that of DA in LD. The m6A-Seq of LD indicated m6A peaks were mainly enriched in the coding sequence (CDS) and stop codon. A total of 161 differentially methylated genes (DMGs) and 1294 differentially expressed genes (DEGs) were identified in two breeds. GO and KEGG analysis showed that DMGs were closely related to cellular metabolism, and most of DMGs were enriched in pathways related to energy metabolism, muscle growth and development, mainly MAPK signaling pathway, Wnt signaling pathway and CGMP-PKG signaling pathway. The DEGs were significantly enriched in actin binding, calcium ion binding, angiogenesis, and other biological processes, and most of them were enriched in PI3K-Akt and CGMP-PKG signaling pathways. Combined analysis of m6A-Seq and RNA-Seq data revealed a negative correlation between differentially methylated m6A levels and mRNA abundance, and mRNA expression of the gene with m6A peak near 3'UTR will decrease. In addition, 11 DMGs regulating cell differentiation, muscle growth and development were identified. This study displayed the m6A profiles and distribution patterns in the goat transcriptome, determined the potential role of m6A modification in muscle growth and provided a new reference for the further study of goat skeletal muscle development.
| S-EPMC10048064 | biostudies-literature
Project description:BackgroundCucumber 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.ResultsWe 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.ConclusionsOur 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.
| S-EPMC8574010 | biostudies-literature
Project description:More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N6-methyladenosine (m6A), have been detected in mRNA, opening the window into the realm of epitranscriptomics. The m6A modification is the most abundant modification in mRNA and non-coding RNA (ncRNA). At the molecular level, m6A affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA (miRNA) maturation, playing essential roles in a range of cellular processes. The m6A modification is regulated by three classes of proteins generally referred to as the "writer" (adenosine methyltransferase), "eraser" (m6A demethylating enzyme), and "reader" (m6A-binding protein). The m6A modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology (YTH) family proteins, selectively bind to RNA and affect its fate in an m6A-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the m6A modification, and provide our insights into the m6A-mediated gene regulation.
| S-EPMC6112310 | biostudies-literature