Sequence- and structure-specific cytosine-5 methylation in mRNAs by NSUN6
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA591625 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10530104.fastq.gz | Fastqsanger.gz | |||
| SRR10530105.fastq.gz | Fastqsanger.gz | |||
| SRR10530106.fastq.gz | Fastqsanger.gz | |||
| SRR10530107.fastq.gz | Fastqsanger.gz | |||
| SRR10530108.fastq.gz | Fastqsanger.gz |
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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
Project description:The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (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, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
| S-EPMC7826283 | biostudies-literature
Project description:mRNA modifications are vital in regulating cellular processes. Beyond N6-methyladenosine (m6A), most other internal mRNA modifications lack dedicated catalytic machinery and are typically introduced by tRNA-modifying enzymes. The distribution and stoichiometry of these modifications on mRNAs remain debated and require further validation. Furthermore, their precise function remains controversial due to the challenges of excluding the intricate combinational effects of tRNA modifications. Here, we biochemically validate that NSUN6, a tRNA structure-dependent methyltransferase, independently catalyzes 5-methylcytidine (m5C) formation with robust activity on mRNA by recognizing the CUCCA motif in a certain stem-loop structure. NSUN6 employs different strategies to recognize tRNA and mRNA substrates. By introducing mutations, we further separate its catalytic capabilities toward mRNA and tRNA revealing that NSUN6 promotes breast cancer cell migration depending on mRNA m5C modification. Mechanistically, a cohort of mRNAs involved in cell migration carries high levels of NSUN6-mediated site-specific m5C modification, thus being stabilized by the preferential binding of m5C readers YBX1 and YBX3. Moreover, introducing a single-site high-level m5C can significantly increase the stability of therapeutic mRNAs in cells. Our findings underscore the pivotal role of m5C-modified mRNAs in promoting breast cancer metastasis and their potential for therapeutic applications.
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Project description:We generated a map of NSUN6-dependent RNA 5 methyl-cytosine in the human transcriptome by applying RNA bisulfite conversion and sequencing to two sets of cell lines (HUES9 and HEK293) where the RNA cytosine 5 methyl-transfearse NSUN6 has been either knocked out by CRISPR Cas9 or overexpressed using a PiggyBac vector. We apply this and other molecular methods to validate NSUN6 RNA methylation targets with single nucelotide resolution in the human transcriptome.
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Project description:mRNA modifications are vital in regulating cellular processes. Beyond N6-methyladenosine (m6A), most other internal mRNA modifications lack dedicated catalytic machinery and are typically introduced by tRNA-modifying enzymes. The distribution and stoichiometry of these modifications on mRNAs remain debated and require further validation. Furthermore, their precise function remains controversial due to the challenges of excluding the intricate combinational effects of tRNA modifications. Here, we biochemically validate that NSUN6, a tRNA structure-dependent methyltransferase, independently catalyzes 5-methylcytidine (m5C) formation with robust activity on mRNA by recognizing the CUCCA motif in a certain stem-loop structure. NSUN6 employs different strategies to recognize tRNA and mRNA substrates. By introducing mutations, we further separate its catalytic capabilities toward mRNA and tRNA revealing that NSUN6 promotes breast cancer cell migration depending on mRNA m5C modification. Mechanistically, a cohort of mRNAs involved in cell migration carries high levels of NSUN6-mediated site-specific m5C modification, thus being stabilized by the preferential binding of m5C readers YBX1 and YBX3. Moreover, introducing a single-site high-level m5C can significantly increase the stability of therapeutic mRNAs in cells. Our findings underscore the pivotal role of m5C-modified mRNAs in promoting breast cancer metastasis and their potential for therapeutic applications.
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Project description:We performed polyribosome-bound mRNAs sequencing (polyribosome-seq) using the samples from NSUN6 depleted K150 cells and control cells and calculated the translation efficiency (TE) for each sample.
2024-03-20 | GSE242287 | GEO
Project description:A cohort of mRNAs undergo high-stoichiometry NSUN6-mediated site-specific m5C modification
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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 downstream G-rich triplet motif and are computationally predicted to be located at the 5' end of putative hairpin structures, are methylated by NSUN2. Type II m5C sites contain a downstream UCCA motif and are computationally predicted to be located in the loops of putative hairpin structures. However, their biogenesis remains unknown. Here we identified NSUN6, a methyltransferase that is known to methylate C72 of tRNAThr and tRNACys, as an mRNA methyltransferase that targets Type II m5C sites. Combining the RNA secondary structure prediction, miCLIP, and results from a high-throughput mutagenesis analysis, we determined the RNA sequence and structural features governing the specificity of NSUN6-mediated mRNA methylation. Integrating these features into an NSUN6-RNA structural model, we identified an NSUN6 variant that largely loses tRNA methylation but retains mRNA methylation ability. Finally, we revealed a weak negative correlation between m5C methylation and translation efficiency. Our findings uncover that mRNA m5C is tightly controlled by an elaborate two-enzyme system, and the protein-RNA structure analysis strategy established may be applied to other RNA modification writers to distinguish the functions of different RNA substrates of a writer protein.
| S-EPMC8288183 | biostudies-literature
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
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