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RNA expression in NSUN6 knockout cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155203 | GEO | 2020/10/29

REPOSITORIES: GEO

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Identifying the impact of NSUN6 mutants on tRNA methylation

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.

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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

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

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

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Genome-wide identification of mRNA 5-methylcytosine in mammals [human_BS]

Project description:We report a robust experimental and computational framework to identify mRNA m5C sites with high specificity. A quantitative atlas of RNA m5C in mammals with five thousand high-confidence exonic m5C sites has been developed based on our framework.

2019-04-18 | GSE122254 | GEO

Genome-wide identification of mRNA 5-methylcytosine in mammals [RIP_seq]

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Genome-wide identification of mRNA 5-methylcytosine in mammals [mouse_BS]

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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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