TRNA 5-methylcytosine in Arabidopsis thaliana
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE68445 | GEO | 2015/10/01
SECONDARY ACCESSION(S): PRJNA282749
REPOSITORIES: GEO
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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 ribosomal RNAs of all three sub-cellular transcriptomes in Arabidopsis thaliana. m5C sites in rRNAs were also anlyzed in Arabidopsis T-DNA knockouts for the RNA methyltransferases TRM4A, TRM4B, TRDMT1, NSUN5, NOP2A, NOP2B and NOP2C.
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Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana seedlings. m5C sites were also analyzed in Arabidopsis trm4b-1 and trdmt1 T-DNA KO mutants for the RNA methyltransferases TRM4B and TRDMT1.
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Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana roots. m5C sites were analyzed in wild type (WT) and an Arabidopsis T-DNA KO mutant for the RNA methyltransferase TRM4B.
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2015-10-01 | GSE68448 | GEO
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 non-coding ribosomal RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba. RNA-seq was performed on total RNA for all species except for Arabidopsis in order to generate rRNA reference sequences using the Arabidopsis rRNA sequences (TAIR10) as a guide.
2015-10-01 | GSE68447 | GEO
Project description:Methylation of carbon 5 in cytosine (5-methylcytosine; m5C) is a well-characterized DNA modification, and is also predominantly reported in highly abundant noncoding RNAs, such as rRNA and tRNA, in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here we develop an m5C RNA immunoprecipitation followed by deep sequencing approach (m5C-RIP-seq) to achieve transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identifies 6,045 putative m5C peaks in 4,465 expressed genes in young seedlings. m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further show that a RNA (cytosine-5)-methyltransferase, tRNA specific methyltransferase 4B (TRM4B), exhibits the m5C mRNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C levels in root mRNA. Furthermore, TRM4B affects transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker widely distributed in plant genes, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.
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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:Methylation of carbon 5 in cytosine (5-methylcytosine; m5C) is a well-characterized DNA modification, and is also predominantly reported in highly abundant noncoding RNAs, such as rRNA and tRNA, in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here we develop an m5C RNA immunoprecipitation followed by deep sequencing approach (m5C-RIP-seq) to achieve transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identifies 6,045 putative m5C peaks in 4,465 expressed genes in young seedlings. m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further show that a RNA (cytosine-5)-methyltransferase, tRNA specific methyltransferase 4B (TRM4B), exhibits the m5C mRNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C levels in root mRNA. Furthermore, TRM4B affects transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker widely distributed in plant genes, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.
2017-10-05 | GSE94003 | GEO
Project description:5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow the m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.
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