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Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [RNA-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: GSE200952 | GEO | 2022/06/16

REPOSITORIES: GEO

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Effects of NSUN6 deficiency by systematic transcriptomes and m5C profiles in mice across tissue [BS-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 | GSE200951 | 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

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

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.

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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

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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

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

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

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Systematic Evaluation of Parameters in RNA Cytosine Methylation Analysis

Project description:Here we systematically analysis bisulfite-treated RNA sequencing data (RNA BS-seq) for the purpose of identifying m5C methylation data from mouse neural stem cells. We isolated mitochondrial fractions to better understand the m5C characteristics of RNAs found in these cellular compartments. Throughout the analysis, we identify key parameters to be used in RNA m5C methylation analysis, and identify downstream effects of artifact detection on RNA m5C levels. In addition, we utilized Unique Molecular Identifiers (UMIs) in conjunction with ERCC (External RNA Controls Consortium) as RNA controls to identify rates of m5C artifcats generated by RNA duplication and PCR amplification error rates. Using various preparation parameters, we identify ideal preparation methods for RNA BS-seq.

2022-04-05 | GSE190614 | GEO

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