Comment[ArrayExpressAccession] E-GEOD-47997 MAGE-TAB Version 1.1 Public Release Date 2013-06-17 Investigation Title Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq] Comment[Submitted Name] Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq] Experiment Description Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally. 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 jacob Solomon Oren Safran Deshet-Unger Akiva Jacob-Hirsch Cesarkas Kabesa Amariglio Unger Rechavi Eyal Person First Name jasmine Oz Shirely Michal Naamit Pinchas Jasmine Karen Reut Ninette Ron Gideon Eran Person Email j-jacob@sheba.health.gov.il Person Affiliation sheba medical center Person Phone 97235302147 Person Address sheba medical center, tel hashomer, ramat gan, Israel, Israel Person Roles submitter Protocol Name P-GSE47997-4 P-GSE47997-1 P-GSE47997-3 P-GSE47997-2 Protocol Description Alignment: Alignment of RNA-seq reads was done using TopHat (Trapnell et al. 2009), a splicing-aware aligner. The reads were mapped to the human reference genome (hg19). Mismatches were not allowed adjacent to the splice junction, and a splice-junction anchor of at least 12 bases was used. The alignment was done with respect to known Ensembl transcripts (using Ensembl62 transcripts GTF file), and unknown splice-junction detection was set to 'off'. Therefore, unknown splice variants could not be detected, and all results shown in this study are based on known variants. Using SAMtools (Li et al.2009), only paired-end reads were retained, where both ends aligned in a proper orientation and with less than 100,000 bases between them. Differential expression (DE) analysis was done using AltAnalyze (Emig et al. 2010; Salomonis et al. 2010) by counting the reads for each constitutive exon in Ensembl genes (Ensembl62) and using AltAnalyze quantile normalization. Alternative splicing analysis: RNA-seq AS analysis was done using AltAnalyze (Emig et al. 2010; Salomonis et al. 2010) based on alignment and junctions obtained by TopHat (Trapnell et al. 2009). Genome_build: hg19 Supplementary_files_format_and_content: HepG2_AS_region: HepG2 reads mapping and splice junctions were resulted from TopHat. Quantile normalization and alternative splicing (AS) analysis were done using AltAnalyze. AltAnlayze AS results format in xls file (from left column to right column)- Ensembl-ID, Splicing-Index,gene symbol,description, exons,regulation_call, junctionID, fold, adjfold, up_exons, down_exons, functional_prediction, uniprot-ens_feature_predictions, peptide_predictions, ens_overlapping_domains, constitutive_baseline_exp, gene-expression-change, transcript_cluster_ID, ensembl_exons, consitutive_probeset(yes/no), exon-region-ID,exon_annotations, Junction_location Supplementary_files_format_and_content: K562_AS_region: K562 reads mapping and splice junctions were resulted from TopHat. Quantile normalization and alternative splicing (AS) analysis were done using AltAnalyze. AltAnalyze AS results format in xls file (from left column to right col column)- Ensembl-ID, Splicing-Index,gene symbol,description,exons, regulation_call, junctionID, fold, adjfold, up_exons, down_exons, functional_prediction, uniprot-ens_feature_predictions, peptide_predictions, ens_overlapping_domains, constitutive_baseline_exp, gene-expression-change, transcript_cluster_ID, ensembl_exons, consitutive_probeset(yes/no), exon-region-ID,exon_annotations, Junction_location Supplementary_files_format_and_content: HepG2_DE_2_fold: HepG2 reads mapping were resulted from TopHat. Quantile normalization and differential expression (DE) analysis were done using AltAnalyze. 2 fold change was taken as cutoff for this list. Format of xls file (from left colum to righ colum) - GeneID, gene_symbol, full_name, avg-ADARkd, avg-control, log_fold-ADARkd_vs_control, fold-ADARkd_vs_control Supplementary_files_format_and_content: K562_DE_2_fold: K562 reads mapping and junctions were resulted from TopHat. Quantile normalization and differential expression (DE) analysis were done using AltAnalyze.2 fold change was taken as cutoff for this list. Format of xls file (from left column to right column) - GeneID, gene_symbol, full_name, avg-ADARkd, avg-control, log_fold-ADARkd_vs_control, fold-ADARkd_vs_control HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). Different types of siRNA were tested, and the one yielding the best ADAR reduction was chosen. The same cells transected with a plasmid containing the scrambled siRNA (SCR) were used as a control. RNA was purified using TRIzol. Libraries were prepared according to Illumina's instructions. Libraries were sequenced on the Genome Analyzer 2x following the manufacturer's protocols. 76 base paired-end reads were sequenced in Illumina GAIIX with two technical replicates for each sample (2 × control and 2 × ADAR KD for HepG2 and K562). HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Beit-Haemek, Israel)). K562 cells were cultured in RPMI 1640 medium (Beit-Haemek, Israel). Both media were supplemented with 10% fetal calf serum (FCS) (Beit-Haemek, Israel), 2 mM glutamine, 100 mg/ml streptomycin and 100 units/ml penicillin (Beit-Haemek, Israel), at 370c in a humidified incubator with 5% CO2. Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CELL LINE TRANSFECTED WITH Experimental Factor Type cell line transfected with Publication Title Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR). Publication Author List Solomon O, Oren S, Safran M, Deshet-Unger N, Akiva P, Jacob-Hirsch J, Cesarkas K, Kabesa R, Amariglio N, Unger R, Rechavi G, Eyal E PubMed ID 23474544 Publication DOI 10.1261/rna.038042.112 Comment[SecondaryAccession] GSE47997 Comment[GEOReleaseDate] 2013-06-17 Comment[ArrayExpressSubmissionDate] 2013-06-17 Comment[GEOLastUpdateDate] 2013-06-21 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE47997_HepG2_AS_regions.xls Comment[AdditionalFile:Data2] GSE47997_HepG2_DE_2_fold.xls Comment[AdditionalFile:Data3] GSE47997_K562_AS_regions.xls Comment[AdditionalFile:Data4] GSE47997_K562_DE_2_fold.xls Comment[SecondaryAccession] SRP026084 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR901895-SRR901902 SDRF File E-GEOD-47997.sdrf.txt