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Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [BS-Seq]

ABSTRACT: we constructed Nsun6-KO mouse, and observed that the wight and appearance of KO mice didn’t show abnormality. Through Bisulfite sequencing (BS-seq) in wild-type (WT) as well as KO mice and mRNA-seq, we identified high confident Nsun6-dependent m5C sites and performed the transcriptome analysis across five tissues.

ORGANISM(S): Mus musculus

PROVIDER: GSE200951 | GEO | 2022/06/16

REPOSITORIES: GEO

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Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [RNA-Seq]

Project description:we constructed Nsun6-KO mouse, and observed that the wight and appearance of KO mice didn’t show abnormality. Through Bisulfite sequencing (BS-seq) in wild-type (WT) as well as KO mice and mRNA-seq, we identified high confident Nsun6-dependent m5C sites and performed the transcriptome analysis across five tissues.

2022-06-16 | GSE200952 | GEO

RNA BS-seq of Nocodazole treated HeLa cells with NSUN family member deficiency

Project description:m5C accumulates on mRNAs in Nocodazole (Noc) treated cells. To figure out which m5C writer is responsible for these m5C sites, we generated three KO cell lines (NSUN2, NSUN5, and NSUN6) with CRISPR induced mutagenesis. We also used siRNA to suppress the expression of Nop2 (NSUN1) in wild-type HeLa cells. Then we performed mRNA BS-seq on the cells harvested after 48 hours Noc-treatment (for siRNA experiments, cells were treated with siRNA for 48 hours first).

2023-08-20 | GSE197650 | GEO

Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [BS-Seq]

Project description:Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [BS-Seq]

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Sequence- and structure-specific cytosine-5 methylation in mRNAs by NSUN6

Project description:Methylation is the most common internal modification in mRNA. While the highly abundant N6-methyladonsine (m6A) modification affects most aspects of mRNA function, the precise functions of the rarer 5-methylcytosine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3’UTR) at the consensus sequence motif CTCCA. Knockout and rescue experiments revealed that only mRNA methylation sites containing the consensus motif depended on the presence of NSUN6. Furthermore, ribosome profiling demonstrated that NSUN6-specific consensus motifs marked translation termination. However, even though NSUN6-methylated mRNAs were reduced in NSUN6 knockout cells, NSUN6 was dispensable for mouse embryonic development. Thus, our study identifies NSUN6 as methyltransferase targeting mRNA in a sequence- and structure-specific manner.

2020-11-20 | GSE140997 | GEO

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

Project description:5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow the m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

2024-07-11 | GSE242724 | GEO

RNA expression in NSUN6 knockout cells

Project description:mRNA m5C, which has recently been implicated in the regulation of mRNA mobility, metabolism, and translation, plays important regulatory roles in various biological events. Two types of m5C sites are found in mRNAs. Type I m5C sites, which contain a 3’G-rich triplet motif and locate in the 5’ end of hairpin structures, are methylated by NSUN2. Type II m5C sites contain a 3’UCCA motif and locate in the loops of hairpin structures. However, their biogenesis remains unknown. Here we identified a novel mRNA methyltransferase that targets Type II m5C sites and generated BS-seq and RNA-seq data to verify our findings.

2020-10-29 | GSE155203 | GEO

Identifying the impact of NSUN6 mutants on tRNA methylation

2020-10-29 | GSE148761 | GEO

Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue

Project description:Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue

| PRJNA827751 | ENA

Genome-wide maps of m5C methylation in neuron of mouse.

Project description:We report the application of RNA bisulfite sequencing(RNA-BS-seq) technology for high-throughput profiling of mouse neuron. Here, we successfully constructed a neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) model,1.5 hours and 3 hours respectively, and obtained an overview of the transcriptome-wide m5C profiles using RNA-BS-seq. We discovered that the distribution of neuronal m5C modifications was highly conserved, significantly enriched in CG-rich regions and concentrated in the mRNA translation initiation regions. After OGD/R, modification level of m5C increased, whereas the number of methylated mRNA genes decreased. The amount of overlap of m5C sites with the binding sites of most RBPs increased significantly, except for that of the RBM3-binding protein. Moreover, hypermethylated genes in neurons were significantly enriched in pathological processes, and the hub hypermethylated genes RPL8 and RPS9 identified by the protein-protein interaction (PPI) network were significantly related to cerebral injury. This study identified novel m5C mRNAs associated with ischemia-reperfusion in neurons, providing valuable perspectives for future studies on the role of the RNA methylation in cerebral ischemia-reperfusion injury.

2021-01-22 | GSE165256 | GEO

RNA m5C Methylation in breast cancer using MeRIP-Seq

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

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