Site-specific cytosine-5 methylation by the mouse RNA methyltransferases Dnmt2 and NSun2 promotes tRNA stability and protein synthesis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE36918 | GEO | 2012/07/04
SECONDARY ACCESSION(S): PRJNA157093
REPOSITORIES: GEO
Similar Datasets
Project description:Mouse embryonic fibroblast (MEFs) cell lines and liver samples from Dnmt2 and Nsun2 single mutant, double mutant and wildtype mice were used to identify potential mRNA substrates of both proteins. MEFs were derived from day 13.5 embryos and immortalized by transfection with a plasmid expressing the SV40 large-T antigen. Total RNA of MEFs cell line at passage 9 and total RNA extracted from 2 months old mouse liver tissues of Dnmt2, Nsun2 single mutant, double mutant and wildtype mice were arrayed on Illumina MouseRef8 v2 chips.
2012-07-03 | E-GEOD-36918 | biostudies-arrayexpress
Project description:Post-transcriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and often organized by the chromatoid body. Chromatoid bodies are large cytoplasmic ribonucleoprotein granules, whose precise function and composition remain unclear. Here, we identify NSun2 as a novel component of the chromatoid body, and further show that this RNA methylase is essential for germ cell differentiation in the mouse testis. Lack of NSun2 leads to down-regulation of genes controlling RNA processing and post-transcriptional repression pathways, including Ddx4, Mili, Piwil1 (Miwi) and Tudor domain containing (Tdrd) proteins. Germ cell differentiation was blocked specifically at the pachytene stage by lack of NSun2, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 with Dnmt2, the only other characterized cytosine-5 RNA methyltransferase to date, indicating that Dnmt2 was not functionally redundant for NSun2 in spermatogonial stem cells or Sertoli cells. Thus, our data indicate that RNA methylation pathways play an essential role in male germ cell differentiation. Four sample groups: Testes from Wild-type and NSun2 knock out mice at 15 and 49 days; 6 samples in each group
2013-02-07 | E-GEOD-39480 | biostudies-arrayexpress
Project description:Post-transcriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and often organized by the chromatoid body. Chromatoid bodies are large cytoplasmic ribonucleoprotein granules, whose precise function and composition remain unclear. Here, we identify NSun2 as a novel component of the chromatoid body, and further show that this RNA methylase is essential for germ cell differentiation in the mouse testis. Lack of NSun2 leads to down-regulation of genes controlling RNA processing and post-transcriptional repression pathways, including Ddx4, Mili, Piwil1 (Miwi) and Tudor domain containing (Tdrd) proteins. Germ cell differentiation was blocked specifically at the pachytene stage by lack of NSun2, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 with Dnmt2, the only other characterized cytosine-5 RNA methyltransferase to date, indicating that Dnmt2 was not functionally redundant for NSun2 in spermatogonial stem cells or Sertoli cells. Thus, our data indicate that RNA methylation pathways play an essential role in male germ cell differentiation.
2013-02-07 | GSE39480 | GEO
Project description:We report development of a mechanism-based technique, Aza-IP, that utilizes the stable covalent linkage formed in vivo between an RNA methyltransferase (m5C-RMT) and 5-azacytidine (5-aza-C) incorporated within the target RNA to enable specific target enrichment, coupled with high-throughput sequencing to provide target identification. We apply Aza-IP to two enzyme types, DNMT2 and NSUN2, the latter with important roles in fertility, intellectual ability, stem cells and cancer. For both, Aza-IP provided >200-fold enrichment of their known human tRNA targets. For NSUN2, we reveal many novel tRNA and non-coding RNA targets, all linked to NSUN2 nucleolar localization, greatly increasing the potential repertoire underlying loss-of-function pathologies. Strikingly, we observe a C>G transversion ‘signature’ specifically at the target cytosine, which enables the identification of the exact target cytosine within the RNA in the same experiment.
2013-04-21 | GSE38957 | GEO
Project description:We report development of a mechanism-based technique, Aza-IP, that utilizes the stable covalent linkage formed in vivo between an RNA methyltransferase (m5C-RMT) and 5-azacytidine (5-aza-C) incorporated within the target RNA to enable specific target enrichment, coupled with high-throughput sequencing to provide target identification. We apply Aza-IP to two enzyme types, DNMT2 and NSUN2, the latter with important roles in fertility, intellectual ability, stem cells and cancer. For both, Aza-IP provided >200-fold enrichment of their known human tRNA targets. For NSUN2, we reveal many novel tRNA and non-coding RNA targets, all linked to NSUN2 nucleolar localization, greatly increasing the potential repertoire underlying loss-of-function pathologies. Strikingly, we observe a C>G transversion ‘signature’ specifically at the target cytosine, which enables the identification of the exact target cytosine within the RNA in the same experiment. The general design of Aza-IP involves nine steps: 1) Expression of an epitope-tagged m5C-RMT derivative (or use of an antibody capable of immuno-precipitating the RNA-bound enzyme), 2) cell growth in the presence of 5-aza-C, which incorporates at low/moderate levels into nascent RNA, 3) cell lysis, 4) immuno-precipitation of the subject m5C-RMT, a portion of which is covalently attached to target RNAs bearing 5-aza-C, 5) stringent washing to remove RNA contaminants, 6) RNA fragmentation, release and purification, 7) ligation of adaptor oligos to the RNA, and the creation of a cDNA library in a manner that enables strand-specific assignments, 8) cDNA sequencing, and 9), mapping and examination of sequence reads to define RNA targets and site of cross-linking/catalysis.
2013-04-21 | E-GEOD-38957 | biostudies-arrayexpress
Project description:Dnmt2 and NSun2 are (cytosine-5) RNA methyltransferases. Using CRISPR/Cas9 we created null mutations in Dnmt2 and NSun2 genes in Drosophila melanogaster. We also ectopically expressed a wild type and catalytically inactive Dnmt2 form in the Dnmt2 mutant background. RNA bisulfite sequencing was used to follow RNA methylation at partical tRNA substrates.
2018-01-16 | GSE109133 | 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: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: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.
2013-07-23 | GSE44385 | 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.
2013-07-23 | GSE44384 | GEO