Comment[ArrayExpressAccession]	E-GEOD-44384		
MAGE-TAB Version	1.1		
Public Release Date	2013-07-23		
Investigation Title	NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs [RNA-seq]		
Comment[Submitted Name]	NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs [RNA-seq]		
Experiment 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		
Term Source Name	ArrayExpress	EFO	
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl	
Person Last Name	Hussain	Hussain	
Person First Name	Shobbir	Shobbir	
Person Email	sh465@cam.ac.uk		
Person Affiliation	Wellcome Trust/Medical Research Council Stem Cell Institute		
Person Address	Wellcome Trust/Medical Research Council Stem Cell Institute, Tennis Court Road, Cambridge, United Kingdom		
Person Roles	submitter		
Protocol Name	P-GSE44384-3	P-GSE44384-1	P-GSE44384-2
Protocol Description	Reads were aligned to the human reference genome (GRCh37/hg19) using tophat (version 2.0.4). Only the reads uniquely aligning to the genome and aligning without mismatch were considered for further analysis. Fragment counts were normalized and differential abundance of fragments was evaluated with the Bioconductor DESeq package (http://www-huber.embl.de/users/anders/DESeq). Genome_build: GRCh37/hg19 Supplementary_files_format_and_content: tab-delimited text files include count values for each sample	HEK293 cells were transfected with siRNAs directed against NSUN2 or otherwise a scrambled control sequence as a negative control. 48 hours post-transfection, cells were harvested.	Total RNA was obtained using the standard Trizol extraction protocol and the mRNA fraction was subsequently purified using oligodT magnetic dynabeads. Libraries were prepared using the Illumina TruSeq mRNA library prep kit.
Protocol Type	normalization data transformation protocol	sample treatment protocol	nucleic acid library construction protocol
Experimental Factor Name	RNA interference		
Experimental Factor Type	RNA interference		
Publication Title	NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs.		
Publication Author List	Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J, Frye M		
PubMed ID	23871666		
Publication DOI	10.1016/j.celrep.2013.06.029		
Comment[SecondaryAccession]	GSE44384		
Comment[GEOReleaseDate]	2013-07-23		
Comment[ArrayExpressSubmissionDate]	2013-02-19		
Comment[GEOLastUpdateDate]	2013-07-24		
Comment[AEExperimentType]	RNA-seq of coding RNA		
Comment[SecondaryAccession]	SRP018786		
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR748515-SRR748522		
SDRF File	E-GEOD-44384.sdrf.txt		
Comment[AEExperimentDisplayName]	Transcription profiling by high throughput sequencing of HEK293 cells transfected with siRNA against Nsun2