Epitranscriptomic maps of 5-methylcytosine reveal substrate diversity of NSUN methyltransferases and links to mRNA translation and turnover
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ABSTRACT:
PROVIDER: PRJNA1060300 | ENA |
REPOSITORIES: ENA
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Project description:Multiple epitranscriptomic maps of the RNA modification 5-methylcytosine (m5C) have been prepared, often diverging markedly from each other in terms of site abundance and identity. Differences in detection methods, data depth and analysis pipelines, but also biological factors underly much of this disparity. To address this, we re-analysed available datasets from five human cell lines and seven tissues, generated by the prevailing bisulfite RNA sequencing method, with a coherent m5C site calling pipeline. We used the resulting union list of 6,393 m5C sites called across the broader transcriptome to study site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed prior observations such as predominance of m5C sites within mRNA exons, enrichment around start codons, and that the tRNA:m5C methyltransferases (MTases) NSUN2 and 6 are the main m5C ‘writers’ for mRNAs. Each NSUN enzyme recognises mRNA features that strongly resemble their respective canonical substrates. Assessing proximity between mRNA m5C sites and footprints of RNA-binding proteins reconfirmed the mRNA export factor ALYREF as an m5C ‘reader’ and identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. Experiments in HeLa cells lacking NSUN2 showed that this affected both, steady-state level and UPF1-binding to target mRNAs. Our studies emphasise the emerging diversity of both, m5C writers and readers, affecting mRNA function.
2024-07-04 | GSE252369 | GEO
Project description:Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.
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Project description:By comparing mRNAs contained in the polysome fraction to total mRNAs in Caenorhabditis elegans exposed to either RNAi control or nsun-1 RNAi, we found that loss of NSUN-1 translationally represses genes associated with collagens, cuticle integrity and development. These changes explain phenotypes of nsun-1 depletion, which include impaired fecundity, gonad extrusion and reduced barrier function.
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Project description:Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in transfer RNAs of all three sub-cellular transcriptomes of Arabidopsis thaliana. 5-methylcytosine sites in tRNAs were also determined in Arabidopsis T-DNA knockouts for the RNA methyltransferases TRM4A, TRM4B, TRDMT1, NSUN5 and NOP2A.
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Project description:Exposure of airborne particulate matter (PM) with an aerodynamic diameter less than 2.5??m (PM2.5) is epidemiologically associated with lung dysfunction and respiratory symptoms, including pulmonary fibrosis. However, whether epigenetic mechanisms are involved in PM2.5-induced pulmonary fibrosis is currently poorly understood. Herein, using a PM2.5-induced pulmonary fibrosis mouse model, we found that PM2.5 exposure leads to aberrant mRNA 5-methylcytosine (m5C) gain and loss in fibrotic lung tissues. Moreover, we showed the m5C-mediated regulatory map of gene functions in pulmonary fibrosis after PM2.5 exposure. Several genes act as m5C gain-upregulated factors, probably critical for the development of PM2.5-induced fibrosis in mouse lungs. These genes, including Lcn2, Mmp9, Chi3l1, Adipoq, Atp5j2, Atp5l, Atpif1, Ndufb6, Fgr, Slc11a1, and Tyrobp, are highly related to oxidative stress response, inflammatory responses, and immune system processes. Our study illustrates the first epitranscriptomic RNA m5C profile in PM2.5-induced pulmonary fibrosis and will be valuable in identifying biomarkers for PM2.5 exposure-related lung pathogenesis with translational potential.
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Project description:Increasing evidence implicates critical roles of various epitranscriptomic RNA modifications in different biological processes. Methyltransferase METTL8 installs 3-methylcytosine modification of mitochondrial tRNAs in vitro, however, its role in intact biological systems is completely unknown. Here we show that Mettl8 is localized in the mitochondria and installs m3C in mitochondrial tRNASer(UCN)/Thr in mouse embryonic cortical neural stem cells. At molecular and cellular levels, Mettl8 deletion in cortical neural stem cells leads to reduced mitochondrial protein translation and attenuated respiration activity. At the functional level, conditional Mettl8 deletion in mice results in impaired embryonic cortical neural stem cell maintenance in vivo, which can be rescued by pharmacologically enhancing mitochondrial functions. Similarly, METTL8 regulates mitochondrial protein expression and neural stem cell maintenance in human forebrain cortical organoids. Together, our study reveals a conserved epitranscriptomic mechanism of Mettl8 and mitochondrial tRNA m3C modification in maintaining embryonic cortical neural stem cells in mice and humans.
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Project description:Total RNA samples from human bronchial epithelial BEAS-2B passage-matched control cells and Cr(VI)-transofmred BEAS-2B cells were submitted to ArraySatr for total RNA m6A epitranscriptomic microarray analysis
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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.
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