Genome-wide maps of m6A circRNAs identify widespread and cell-type-specific methylation patterns that are distinct from mRNAs
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE85324 | GEO | 2017/08/29
SECONDARY ACCESSION(S): PRJNA338243
REPOSITORIES: GEO
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Project description:N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic RNA. Recent mapping of m6A methylomes in mammals, yeast, and plants as well as characterization of m6A methyltransferases, demethylases, and binding proteins have revealed regulatory functions of this dynamic RNA modification. In bacteria, although m6A is present in ribosomal RNA (rRNA), its occurrence in messenger RNA (mRNA) still remains elusive. Here, we used liquid chromatography-mass spectrometry (LC-MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved distinct m6A pattern that is significantly different from that in eukaryotes. Most m6A peaks are located inside open reading frames (ORF), and carry a unique consensus motif (GCCAU). Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified transcripts are associated with respiration, amino acids metabolism, stress response, and small RNAs genes, suggesting potential regulatory roles of m6A in these pathways. m6A profiling in E.coli and P.aeruginosa mRNA
2015-07-08 | E-GEOD-65347 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.
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Project description:Severe acute pancreatitis (SAP) is the most serious type of pancreatitis with high morbidity and mortality. The underlying pathophysiological mechanism of SAP is complicated and lacking in effective therapeutic options in clinic. In recent years, circular RNAs (circRNAs) are found to be N6-methyladenosine (m6A)-modified and m6A modification of circRNAs plays important roles in physiological and pathological processes. However, the role of m6A modification of circRNAs in SAP remains unknown. Here, we aim to identify differentially expressed m6A circRNAs in SAP and to determine their biological significance and potential mechanisms in SAP. Firstly, we identified 903 m6A peaks that distribute on 781 circRNAs in SAP and control groups. Among them, 57 circRNAs with differentially expressed m6A peaks were identified, of which 32 were upregulated and 25 were downregulated. The total m6A level of circRNAs was reduced compared with control group. Moreover, the function analysis of these m6A circRNAs in SAP found that some important pathways involved in the pathogenesis of SAP, such as protein digestion and regulation of autophagy. In m6A circRNA-microRNA networks, several important miRNAs involved in the occurrence and development of SAP were found to bind to these m6A circRNAs potentially, such as miR-24-3p, miR-26a, miR-92b, miR-216b, miR-324-5p and miR-762. Notably, ALKBH5 was found to be upregulated in SAP. In conclusion, these results demonstrated that m6A modification of circRNAs was widely existed and may play important roles in the pathogenesis of SAP. Our findings provide novel insights regarding understanding the pathophysiological mechanism of SAP and seeking new therapeutic targets for SAP.
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Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates
2016-06-24 | E-GEOD-66086 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We present eIF3a PAR-iCLIP data which demonstrate that eIF3 predominantly binds mRNAs within the 5'UTR. eIF3 binding sites are also in proximity to m6A residues within the 5'UTR of cellular mRNAs. Two replicates of eIF3a PAR-iCLIP in HEK293T cells.
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Project description:N6-Methyladenosine (m6A) is well-known for controlling different processes of linear RNA metabolism. Conversely, its role in the biogenesis and function of circular RNAs (circRNAs) is still poorly understood. Here, we characterize circRNA expression in the pathological context of rhabdomyosarcoma (RMS), observing a global increase when compared to wild-type myoblasts. For a set of circRNAs, such an increase is due to the raised expression of the m6A machinery, which we also find to control the proliferation activity of RMS cells. Furthermore, we identify the RNA helicase DDX5 as a novel mediator of the back-splicing reaction and as a co-factor of the m6A regulatory network. DDX5 and the m6A reader YTHDC1 are shown to interact and to promote the production of a common subset of circRNAs in RMS. In line with the observation that YTHDC1/DDX5 depletion reduces RMS proliferation, our results provide proteins and RNA candidates for the study of rhabdomyosarcoma tumorigenicity.
2023-03-20 | GSE207453 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate and function. Current m6A mapping approaches rely on immunoprecipitation of m6A-containing RNA fragments to identify regions of transcripts that contain m6A. This approach localizes m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here we show that anti-m6A antibodies can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Similarly, we find these antibodies induce mutational signatures at N6, 2’-O-dimethyladenosine (m6Am), a nucleotide found at the first encoded position of certain mRNAs. Using these mutational signatures, we map m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identify snoRNAs as a novel class of m6A-containing ncRNAs. UV-crosslinking and immunoprecipitation with m6A-specific antibodies was used to map m6A and m6Am in cellular RNA with single nucleotide resolution.
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Project description:To explore the involvement of N6-methyladenosine (m6A) modification in circular RNAs (circRNAs) and relevant methyltransferases in the lesion of lens epithelium cells (LECs) under the circumstances of age-related cataract (ARC).
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Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
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