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Simultaneous identification of m6A and m5C reveals coordinated RNA modification at single-molecule resolution [mouse frontal cortex]

ABSTRACT: The expanding field of epitranscriptomics might rival the epigenome in the diversity of biological processes impacted. However, the identification of multiple modification types in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual transcript molecules in a single condition as well as differential methylation between two conditions, using nanopore signals. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. Moreover, CHEUI’s unique capability to identify different modifications in the same signal data reveals a non-random co-occurrence of m6A and m5C in transcripts in human cell lines and during mouse embryonic brain development. CHEUI unlocks the capability of studying links between multiple RNA modifications and phenotypes, enabling the discovery of new epitranscriptome functions. Furthermore, CHEUI's training and testing protocols are adaptable to other modifications, making it a versatile RNA technology.

ORGANISM(S): Mus musculus

PROVIDER: GSE211760 | GEO | 2022/10/31

REPOSITORIES: GEO

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Simultaneous identification of m6A and m5C reveals coordinated RNA modification at single-molecule resolution [HeLa cells]

Project description:The expanding field of epitranscriptomics might rival the epigenome in the diversity of biological processes impacted. However, the identification of multiple modification types in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual transcript molecules in a single condition as well as differential methylation between two conditions, using nanopore signals. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. Moreover, CHEUI’s unique capability to identify different modifications in the same signal data reveals a non-random co-occurrence of m6A and m5C in transcripts in human cell lines and during mouse embryonic brain development. CHEUI unlocks the capability of studying links between multiple RNA modifications and phenotypes, enabling the discovery of new epitranscriptome functions. Furthermore, CHEUI's training and testing protocols are adaptable to other modifications, making it a versatile RNA technology.

2022-10-31 | GSE211759 | GEO

Simultaneous identification of m6A and m5C reveals coordinated RNA modification at single-molecule resolution[bisulfite RNA sequencing of synthetic constructs]

2024-01-16 | GSE253150 | GEO

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Project description:Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC), RNA-protein enrichment, and quantitative proteomics. We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and use quantitative nucleoside LC-MS to show that ALKBH1 is the major hm5C and f5C-forming enzyme in RNA, including upon polyadenylated RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-based iCLIP analysis to show that ALKBH1 oxidizes m5C in a variety of tRNA anticodon stem loops (ASL), as well as on mRNA and lncRNA, and analyze its substrate specificity using in vitro enzymatic assays. Finally, we apply pyridine borane-mediated sequencing to identify f5C sites in tRNA ASLs. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

2022-07-16 | PXD029955 | Pride

Mapping of m6A and m5C on MLV with PA-antibody-seq

Project description:While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N6-methyladenosine (m6A), 5-methylcytosine (m5C) and 2’O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.

2019-06-01 | GSE129302 | GEO

Mapping the m1A, m5C, m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP-seq

Project description:The epigenetic modifications play important regulatory roles in tissue development, maintenance of physiological functions and pathological process. RNA methylations, including newly identified m1A, m5C, m6A and m7G, are important epigenetic modifications. However, how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study, we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A, m5C, m6A and m7G methylation of mRNA, we found that within the whole brain transcriptome, with the increase of the gene expression levels, the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia-treated zebrafish brains, the proportion of m1A is decreased, affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A, m5C, m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A, m5C, m6A and m7G in the regulation of miRNA and repeat elements in vertebrates, and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome.

2022-01-26 | GSE194284 | GEO

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators II

Project description:Cellular RNA is decorated with over 160 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that could modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq).

2020-11-04 | GSE157367 | GEO

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators I

2020-11-04 | GSE157366 | GEO

Transfer learning enables identification of multiple types of RNA modification using nanopore direct RNA sequencing

Project description:RNA internal modifications play critical role in development of multicellular organisms and their response to environmental cues. Using nanopore direct RNA sequencing (DRS), we constructed a large in vitro epitranscriptome (IVET) resource from plant cDNA library labeled with m6A, m1A and m5C respectively. Furthermore, after transfer learning, the pre-trained model was used to detect additional RNA internal modification such as m1A, hm5C, m7G and Ψ modification. Finally, we illustrated a global view of epitranscriptome with m6A, m1A, m5C, m7G and Ψ modification in rice seedlings under normal and high salinity environment. In summary, we provided a strategy for creating IVET resource from cDNA library and developed a computational method that use IVET-based transfer learning termed TandemMod for profiling epitranscriptome landscape with co-occupancy of multiple types of RNA modification in plants responsive to environmental signal.

2024-03-28 | GSE227087 | GEO

Overview of distinct 5-methylcytosine profiles of messenger RNA in normal and knock-down NSUN2 colorectal cancer cells

Project description:Background: Colorectal cancer (CRC) is a harmful cancer with high morbidity and poor prognosis. There is growing evidence that RNA methylation is closely related to the occurrence of cancer and its malignant biological behavior. N6-methyladenosine (m6A) methylation is the most common RNA modification in eukaryotes, and its multiple regulatory mechanisms in CRC have been elucidated from multiple perspectives. At the same time, the role of 5-methylcytosine (m5C), another important and widely distributed methylation modification, in CRC is far from being elucidated. Methods: In this study, we used RNA immunoprecipitation sequencing combined with bioinformatics methods to identify the m5C peaks on messenger RNA (mRNA) in HCT15 cells and sh-NSUN2 HCT15 cells, understand which transcripts are modified by m5C, and characterize the distribution of m5C modifications. In addition, we performed further bioinformatics analysis of the detected data to initially clarify the potential function of these m5C-modified transcripts. Results: We found significant differences in the distribution of m5C between HCT15 cells and sh-NSUN2 HCT15 cells, suggesting that m5C is likely to play a key role in the occurrence and development of CRC. Furthermore, Gene Ontology (GO) enrichment analysis showed that genes altered by m5C were mainly enriched in phylogeny, synaptic membrane, and transcription factor binding.The Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis showed that the genes altered by m5C are enriched in ECM receptor interaction pathway, the circadian pathway, and the cAMP signaling pathway. Conclusions: Here, our study preliminarily revealed the different distribution patterns of m5C between HCT15 cell and sh-NSUN2 HCT15 cell. Our results open a new window to understand the role of m5C RNA methylation of mRNA in the development of CRC.

2023-03-02 | GSE226129 | GEO

Systematic calibration of epitranscriptomic atlas using a synthetic modification-free RNA library

Project description:Recent years has witnessed rapid progress of the field epitranscriptomics. Functional interpretation of epitranscriptome relies on mapping technologies which determine the localization and stoichiometry of various RNA modifications. However, contradictory results are derived from different studies, questioning the biological impacts of certain RNA modifications. Here, we develop an approach for the generation of synthetic RNA library resembling the endogenous transcriptome but lacking modifications. Incorporating this modification-free RNA library as a negative control into established techniques, we obtain precise and quantitative maps of m6A and m5C after removing the pervasive false positives resulted from other elements such as specific sequence context and RNA secondary structure.

2021-08-18 | GSE151028 | GEO

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