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Transcriptome-wide mapping of m6A in bovine oocytes and preimplantation embryos by SAC-seq

ABSTRACT: RNA N6-methyladenosine (m6A) plays a crucial role in regulating gene expression and early embryonic development. However, the m6A dynamics at single-nucleotide resolution during preimplantation development has not been characterized, and the function of m6A marks in specific transcripts remain largely unexplored. Here, by using SAC-seq, we overcome the limitation of antibody-based m6A profiling and define m6A landscapes in bovine oocytes and pre-implantation embryos at unprecedented level. Notably, we identify a specific m6A site on RPL12 (m6A-RPL12), which was highly methylated at the 8-cell stage. m6A-RPL12 deficient leads to reduced protein synthesis, disrupted expression of translation-related genes, and impaired zygotic genome activation and blastocyst formation. Supplementation with wild-type RPL12 mRNA failed to rescue the developmental arrest, indicating that m6A regulation extends beyond transcript abundance. This study provides invaluable m6A single-nucleotide resolution recourse in early embryogenesis and demonstrates that precise regulation of m6A at selected transcripts can shape early developmental outcomes in mammals.

ORGANISM(S): Bos taurus

PROVIDER: GSE301330 | GEO | 2026/06/09

REPOSITORIES: GEO

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Transcriptome-wide mapping of m6A in bovine oocytes and preimplantation embryos by SAC-seq [SAC-Seq]

Project description:RNA N6-methyladenosine (m6A) plays a crucial role in regulating gene expression and early embryonic development. However, the m6A dynamics at single-nucleotide resolution during preimplantation development has not been characterized, and the function of m6A marks in specific transcripts remain largely unexplored. Here, by using SAC-seq, we overcome the limitation of antibody-based m6A profiling and define m6A landscapes in bovine oocytes and pre-implantation embryos at unprecedented level. Notably, we identify a specific m6A site on RPL12 (m6A-RPL12), which was highly methylated at the 8-cell stage. m6A-RPL12 deficient leads to reduced protein synthesis, disrupted expression of translation-related genes, and impaired zygotic genome activation and blastocyst formation. Supplementation with wild-type RPL12 mRNA failed to rescue the developmental arrest, indicating that m6A regulation extends beyond transcript abundance. This study provides invaluable m6A single-nucleotide resolution recourse in early embryogenesis and demonstrates that precise regulation of m6A at selected transcripts can shape early developmental outcomes in mammals.

2026-06-10 | GSE301371 | GEO

Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome

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.

2015-06-29 | E-GEOD-63753 | biostudies-arrayexpress

High-resolution mapping reveals a conserved, widespread, dynamic meiotically regulated mRNA methylation program

Project description:N6-methyladenosine (m6A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m6A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated 8/8 methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time-course, and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminates a conserved, dynamically regulated methylation program in yeast meiosis, and provides an important resource for studying the function of this epitranscriptomic modification. Examination of m6A methylation under various conditions

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Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome

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SAC-seq for transcriptome-wide quantitative sequencing of mRNA m6A at base resolution

Project description:Here, we reported transcriptome-wide m6A modification maps within single-base resolution using m6A-SAC-seq in rice and Arabidopsis. Our analysis uncovered a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis.

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SAC-seq for transcriptome-wide quantitative sequencing of mRNA m6A at base resolution in Arabidopsis

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The bladder cancer m6A landscape is defined by global methylation dilution and focal 3′-UTR hypermethylation

Project description:N6-Methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNAs and regulates target transcripts throughout the mRNA life cycle. Although changes in m6A have been reported in human cancers, technical limitations have hindered a comprehensive understanding of the cancer-associated m6A landscape. Here, we used GLORI-sequencing to establish the first transcriptome-wide, single-nucleotide resolution maps of m6A in cancer. Differentially methylated transcripts were enriched in oncogenic pathways relevant to urothelial carcinoma of the bladder (UCB). We discovered two key m6A signatures in UCB: a global loss of methylation and a local hypermethylation at 3′-UTRs. Integration of RNA-sequencing data revealed that the global loss resulted from dilution of methylation marks due to increased expression of unmethylated transcripts and decreased expression of highly methylated transcripts. In contrast, local 3′-UTR hypermethylation was associated with the overexpression of VIRMA, a component of the m6A writer complex, which was linked to UCB progression. Our study is the first to describe the m6A epitranscriptomic landscape of cancer at single-base resolution and provides first insights into the processes that generate its characteristic signatures.

2025-07-18 | GSE281749 | GEO

The bladder cancer m6A landscape is defined by global methylation dilution and focal 3′-UTR hypermethylation

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High-resolution mapping reveals a conserved, widespread, dynamic meiotically regulated mRNA methylation program [Mm]

Project description:N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life cycle, but little is known about the pathways regulating this process and its physiological role. Here, we used mass-spectrometry to identify a dense network of proteins physically interacting with METTL3, a core component of the methyltransferase complex, and show that two of them, WTAP and KIAA1429, are required for methylation. Combining high resolution m6A-Seq with knockdown of WTAP allowed us to define accurate maps, at near single-nucleotide resolution, of sites of mRNA methylation across four dynamic programs in human and mouse, including development, differentiation, reprogramming and immune response. Internal WTAP-dependent methylation sites were largely static across the different surveyed conditions and present in the majority of mRNAs. However, methylations were found at much lower levels within highly expressed mRNAs, and methylation is inversely correlated with mRNA stability, consistent with a role in establishing an overall basal, cell-type invariant, distribution of degradation rates. In addition, we identify thousands of WTAP-independent methylation sites at transcription initiation sites, forming part of the mRNA cap structure. We show that the methylations occur at the first transcribed nucleotide, and find that thousands of transcripts are present in different isoforms differing in the methylation state of the first transcribed nucleotide, a previously unappreciated complexity of the transcriptome. Together, our data sheds new light on the proteomic and transcriptional underpinnings of this epitranscriptomic modification in mammals. Examination of m6A methylation across different knockdowns using shRNAs in mouse embryonic fibroblasts, in embyronic and adult brains, and in dendritic cell stimulated with LPS.

2014-07-18 | E-GEOD-55575 | biostudies-arrayexpress

m6A-SAC-seq for quantitative whole transcriptome m6A profiling

Project description:This protocol describes a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The method generates highly reproducible results containing 31,233-129,263 sites using as low as 2 ng of poly-A RNA.

2022-06-30 | GSE198246 | GEO

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