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
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
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.
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
Project description:NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs
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
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.
Project description:<p>Mutations in the cytosine-5 RNA methyltransferase NSun2 can cause neurodevelopmental disorders and symptoms commonly found in patients with Dubowitz-like syndrome. Some tRNAs are known to be methylated NSun2, however the occurrence of cytosine-5 methylation (m<sup>5</sup>C) in other RNA biotypes is still under debate. Location in RNA and function of m<sup>5</sup>C has not been studied yet. This study is aimed at identifying new m<sup>5</sup>C methylated RNA biotypes, as well as the location at specific structures or sequences and the ultimate biological function. The impact of the loss of NSun2-mediated methylation is also determined by comparing gene expression data with the global cytosine-5 RNA methylome in Dubowitz-like syndrome patients. We also use ribosomal profiling to assess the impact of the loss and rescue of Nsun2-mediated cytosine-5 RNA methylation on translation rates and ribosome occupancy.</p>
Project description:Purpose: High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, such those derived from tRNAs. Also recent advances in high-throughput RNA sequencing has revealed the complex RNA modification landscape and the complex role these nucleosides modifactions have in cell signalling, stem cell biology and development. The goal of this study is to establish how tRNA-derived small RNAs can affect stem cell function. Methods: four replicates of tRNAs sequencing of wild-type (WT) and NSun2 -/- mouse embryonic frontal lobes at E18.5 were generated by deep sequencing, using Illumina HiSeq platform. Results: Our analyses reveal that deletion of the cytosine-5 tRNA methylase NSUN2 increases tRNA cleaveage of NSUN2 tRNA substrates.