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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE140997 | GEO | 2020/11/20

REPOSITORIES: GEO

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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.

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Distinct NSun methylases control cytosine-5 methylation of messenger, mitochondrial and transfer RNA: implications for human diseases [miCLIP & Bisfulfite-Seq HEK293]

Project description:Aberrant post-transcriptional methylation is implicated in a wide range of human diseases, yet the specific enzymes responsible for site- and substrate-specific methylation remain largely unknown. Here, we use our recently developed catalysis-dependent RIP sequencing approach (miCLIP) and RNA bisulfite sequencing to map C5-methylcytosine (m5C) in the human transcriptome. We identified two novel m5C methylases NSun3 and NSun6, both of which have strong substrate specificity. In contrast to the previously characterized NSun2, which displays some preference to methylating transfer RNAs (tRNA), NSun6 predominantly targeted 3’ UTRs of messenger RNAs (mRNA), and NSun3 mainly methylated mitochondrial RNAs (mtRNA). Consistent with the miCLIP-predicted RNA target specificity, whole exome sequencing identified NSUN3 loss-of-function mutations in a patient presenting with combined respiratory chain complex deficiency. Functional studies of the patient fibroblast cell line revealed that loss of the NSun3 protein resulted in severe mitochondrial translation defects, which were rescued by expression of wild-type NSun3. In summary, our results reveal how highly conserved NSun m5C methylases partition their substrate specificities to remodel the sequences of particular ribonucleotide classes.

2017-02-14 | GSE66011 | GEO

Distinct NSun methylases control cytosine-5 methylation of messenger, mitochondrial and transfer RNA: implications for human diseases [Bisulfite-Seq dermal fibroblasts]

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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.

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

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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

OsNSUN2-mediated 5-methylcytidine mRNA modification enhances rice adaptation to high-temperature

Project description:Heat stress is one of the extreme weather disasters potently decreasing cereal plant production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in thermoresponse in plants. However, how 5-methylcytosine (m5C) RNA modification contribute to heat stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as the mRNA m5C methyltransferase in rice. The osnsun2 mutants specifically displayed severe phenotypes of temperature- and light-dependent lesion mimic and heat stress hypersensitivity. Heat stress enhanced mRNAs m5C methylation involving photosynthesis and detoxification systems, such as β-OsLCY, OsHO2, OsPAL1, and OsGLYI4, in an OsNSUN2-dependent manner, which then increased the protein synthesis. Consistently, the photosystem of the osnsun2 mutants was vulnerable to high ambient temperature and failed to be repaired under tolerable heat stress. Furthermore, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.

2020-04-15 | PXD017187 | Pride

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