Dynamics of RNA-m5C Modification during Brain Development
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE207092 | GEO | 2022/08/11
REPOSITORIES: GEO
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Project description:RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.
2014-02-20 | E-GEOD-53370 | biostudies-arrayexpress
Project description:Here we report m5C-TAC-seq, a base-resolution sequencing method to directly detect m5C sites without affecting unmodified C. In m5C-seq, we combined TET-mediated oxidation of RNA m5C to f5C with the selective chemical labeling reaction of f5C, enabling both pre-enrichment of m5C-containing RNA and a m5C-to-T transition in reverse transcription. m5C-seq identifies 2,500 sites in the transcriptome of HeLa and 768 sites in the transcriptome-wide of HEK293T. In addition, taking advantage of barcoding and pooling strategy, m5C-seq detected differential m5C sites upon specific methyltransferases depletion in mESCs and dynamically regulated m5C sites under cell fate transition. Moreover, we also detected 215 sites in chromatin-associated RNAs, demonstrating that portion of m5C sites can be co-transcriptionally catalyzed and the existence of m5C methylations in repeat RNAs.
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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.
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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.
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Project description:Dynamics of RNA-m5C Modification during Brain Development
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Project description:Cytosine-5 methylation (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant non-coding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially upregulated, leading to an increased level in unshielded 7SL RNA in cytoplasm, which served as direct ligands for the RIG-I mediated IFN response. In NSUN2 depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating m5C methylome of ncRNAs and their expression.
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Project description:5-methylcytosine (m5C) modification ubiquitously occurs on mammalian mRNAs and plays important roles in multiple biological processes. Currently, the role of m5C in cancer initiation and progression is gaining more and more research attention, but the detailed mechanism remains unclear. As an m5C methyltransferase with unique mRNA catalytic activity, NSUN2 was found to be highly expressed in multiple cancers including gastric, bladder, gallbladder, and breast cancers, suggesting NSUN2 might exert oncogenic properties through affecting m5C levels in cancer cells. Therefore, to understand the potential roles of RNA m5C modification in tumorigenesis of cervical cancer, we established NSUN2 stable knockdown CaSki cell, and perform RNA-seq and RNA-BisSeq to figure out the possible pathway involved in cervical cancer development.
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Project description:The RNA methyltransferase Aly/REF export factor (ALYREF) is considered one type of “reader” protein located in the nucleus that recognizes and binds directly with m5C sites in RNA and facilitates the export of RNA from the nucleus to the cytoplasm. Notably, ALYREF is considered a promising target for diagnosis and prognosis prediction. However, until now, the low number of related studies has limited the understanding of the mechanism of the HCC-promoting effects of ALYREF. To further elucidate the oncogenic roles of ALYREF in hepatocellular carcinoma (HCC), we assessed the expression levels of ALYREF in clinical samples and HCC cell lines and explored the effects of ALYREF deficiency by both in vitro experiments and m5C-methylated RNA immunoprecipitation sequencing (m5C-MeRIP-Seq)
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Project description:Our paper presents the results of a study in which we used whole genome bisulfite sequencing (WGBS) and RNA-Seq (i.e. transcriptomics) to examine the long-term epigenomic dynamics of an experimenta evolution study under high CO2 in the marine cyanobacterium Trichodesmium. We identify m5C methylated sites that rapidly change in response to short-term high CO2 exposure, which are then maintained for 4.5 years even after adaptation (i.e. trait canalization). After 7 years of CO2 selection, high-CO2 triggered methylation levels return to ancestral, low-CO2 levels, consistent with genetic assimilation theory and observations in eukaryotic model systems. These data suggest a potential role for m5C methylation in prokaryotic trait canalization and identify genetic assimilation as an evolutionary mechanism of potential biogeochemical importance under global change factors.
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