Ontology highlight
ABSTRACT:
PROVIDER: PRJNA843487 | ENA |
REPOSITORIES: ENA
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Project description:5-methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. We obtained ~1,000 candidate m5C sites in the wider transcriptome, most of which were found in exonic regions of mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCap and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in mRNA and noncoding RNA. Candidate m5C sites in mRNAs are enriched in 5’UTRs and near start codons, and commonly embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that non-conversion levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Altogether, these findings emphasise the major role of NSUN2 in making this mark transcriptome-wide and further substantiate a functional interdependence of cytosine methylation level with mRNA translation
2020-03-09 | GSE140995 | GEO
Project description:Autosomal-recessive loss of the NSUN2 gene has been recently identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNA). Whether NSun2 methylates additional RNA species is currently debated. Here, we adapted the individual-nucleotide resolution UV cross-linking and immunoprecipitation method (iCLIP) to identify NSun2-mediated methylation in RNA transcriptome. We confirm site-specific methylation in tRNA and identify messenger and non-coding RNAs as potential methylation targets for NSun2. Using RNA bisulfite sequencing we establish Vault non-coding RNAs as novel substrates for NSun2 and identified six cytosine-5 methylated sites. Furthermore, we show that loss of cytosine-5 methylation in Vault RNAs causes aberrant processing into argonaute-associating small RNA fragments (svRNA). Thus, impaired Vault non-coding RNA processing may be an important contributor to the etiology of NSUN2-deficieny human disorders. mRNA-seq in Embryonic kidney (HEK293) cells transfected with siRNA against Nsun2 vs control
2013-07-23 | E-GEOD-44384 | biostudies-arrayexpress
Project description:Mutations in the cytosine-5 RNA methyltransferase NSun2 can cause Intellectual Disability (ID) and symptoms commonly found in patients with Dubowitz syndrome. By analysing gene expression data with the global cytosine-5 RNA methylome in NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the fragmentation of transfer RNAs (tRNA) leading to an accumulation of 5M-bM-^@M-^Y halves. Cleavage of tRNAs by Angiogenin is a common cellular stress response to silence translational programmes, and we show that Angiogenin binds tRNAs lacking site-specific NSun2-methylation with higher affinity. Furthermore, cells lacking functional NSun2 up-regulate stress markers, and deletion of NSun2 compromises cellular survival in response stress stimuli including UV-light and oxidative stress. The decreased tolerance of NSun2 null cells towards oxidative stress can be rescued through inhibition of Angiogenin. In conclusion, cytosine-5 RNA methylation is an essential post-transcriptional mechanism during cellular stress responses and NSun2-mediated tRNA methylation protects from Angiogenin-dependent stress-induced RNA cleavage. RNA Methylation profiling by high throughput sequencing small non-coding RNA profiling by high throughput sequencing Pol III Chromatin-IP profiling by high throughput sequencing
2014-11-06 | E-GEOD-44746 | biostudies-arrayexpress
Project description:Background: As a widespread post-transcriptional RNA modification, N5-methylcytosine (m5C) is implicated in a variety of cellular responses and processes that regulate RNA metabolism. Despite this, a clear understanding of m5C modification’s role and mechanism in angiogenesis is still lacking. Methods: Single-cell RNA sequencing data was analyzed to determine expression of m5C methylase NSUN2. m5C levels were determined by mRNA isolation and anti-m5C dot blot in both hypoxia-induced endothelial cells (ECs) and laser-induced choroidal neovascularization (CNV). In addition, endothelial cell and endothelium‐specific NSUN2‐knockout mouse model were used to investigate the effect of NSUN2 silence on angiogenic phenotype. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5C-methylated RNA immunoprecipitation sequencing (m5C-meRIP-seq). CUT&Tag sequencing was performed to test the histone lactylation signal in the promoter region of NSUN2. Finally, AAV-mediated short hairpin RNAi knockdown of NSUN2 gene expression (AAV-shNSUN2) was constructed to investigate the effect of inhibiting CNV. Results: First, we discovered that m5C methylase NSUN2 expression level and mRNA m5C level were significantly higher in CNV-ECs than in normal ECs. NSUN2 knockdown in ECs inhibited proliferative, migration, and tube formation activities of ECs. Moreover, compared with EC NSUN2flox/flox mice, EC-specific NSUN2-deficient (EC NSUN2-/-) mice displayed less retinal vascular leakage after laser induction. Through multiomics analyses, we subsequently identified A-kinase anchoring protein 2 (AKAP2), a scaffolding protein which isolate Protein kinase A (PKA) to specific subcellular locations through binding to its regulatory subunit, as a downstream candidate target of NSUN2 in ECs. Overexpression of exogenous AKAP2 markedly reversed the inhibitory phenotypes in NSUN2-deficient ECs. Interestingly, laser induced NSUN2 up-regulation was driven by lactate-mediated lactylation on histone H3K18. In CNV models, AAV-mediated repression of NSUN2 modulated highly retinal vascular leakage and choroidal thickness. Conclusion: Overall, our findings indicate that NSUN2 is a novel therapeutic target for choroidal neovascularization.
2024-05-19 | GSE267661 | GEO
Project description:Mutations in the cytosine-5 RNA methyltransferase NSun2 can cause Intellectual Disability (ID) and symptoms commonly found in patients with Dubowitz syndrome. By analysing gene expression data with the global cytosine-5 RNA methylome in NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the fragmentation of transfer RNAs (tRNA) leading to an accumulation of 5’ halves. Cleavage of tRNAs by Angiogenin is a common cellular stress response to silence translational programmes, and we show that Angiogenin binds tRNAs lacking site-specific NSun2-methylation with higher affinity. Furthermore, cells lacking functional NSun2 up-regulate stress markers, and deletion of NSun2 compromises cellular survival in response stress stimuli including UV-light and oxidative stress. The decreased tolerance of NSun2 null cells towards oxidative stress can be rescued through inhibition of Angiogenin. In conclusion, cytosine-5 RNA methylation is an essential post-transcriptional mechanism during cellular stress responses and NSun2-mediated tRNA methylation protects from Angiogenin-dependent stress-induced RNA cleavage.
2014-11-06 | GSE44746 | GEO
Project description:Autosomal-recessive loss of the NSUN2 gene has been recently identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNA). Whether NSun2 methylates additional RNA species is currently debated. Here, we adapted the individual-nucleotide resolution UV cross-linking and immunoprecipitation method (iCLIP) to identify NSun2-mediated methylation in RNA transcriptome. We confirm site-specific methylation in tRNA and identify messenger and non-coding RNAs as potential methylation targets for NSun2. Using RNA bisulfite sequencing we establish Vault non-coding RNAs as novel substrates for NSun2 and identified six cytosine-5 methylated sites. Furthermore, we show that loss of cytosine-5 methylation in Vault RNAs causes aberrant processing into argonaute-associating small RNA fragments (svRNA). Thus, impaired Vault non-coding RNA processing may be an important contributor to the etiology of NSUN2-deficieny human disorders. Identification of Nsun2 targets by miCLIP in Embryonic kidney (HEK293) cells
2013-07-23 | E-GEOD-44385 | biostudies-arrayexpress
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