Mapping of m6A and m5C on HIV with PA-antibody-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA542150 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9036390.fastq.gz | Fastqsanger.gz | |||
| SRR9036391.fastq.gz | Fastqsanger.gz | |||
| SRR9036392.fastq.gz | Fastqsanger.gz | |||
| SRR9036393.fastq.gz | Fastqsanger.gz | |||
| SRR9036394.fastq.gz | Fastqsanger.gz |
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Project description:In this GEO submission we include the PA-m6A-seq and PA-m5C-seq datasets for HIV in CEM & 293T cells
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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.
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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.
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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.
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Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing. Identification of m6A modified sequences in HepG2 cells.
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Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing Identification of m6A modified sequences in mouse liver and human brain
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Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing. Identification of m6A modified sequences in HepG2 cells. HepG2 cells were incubated with either IFNg (200ng/ml) or HGF/SF (10 ng/ml) over night. Stress effects were tested in HepG2 cells by either 30 minutes incubation at 43M-BM-:C (heat shock) or UV irradiation of 0.04 J/cm2 followed by 4 hours of recovery in normal growing conditions prior to harvesting using Trypsin.
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