Comment[ArrayExpressAccession] E-GEOD-41432 MAGE-TAB Version 1.1 Public Release Date 2013-10-01 Investigation Title Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome Comment[Submitted Name] Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome Experiment Description Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes aberrant transcripts arising from mutations and transcriptional mistakes. Moreover, differential transcript processing such as alternative precursor mRNA splicing (AS) can generate NMD substrates, however, the extent of coupled AS and NMD remained unclear. To investigate NMD targeting of AS variants, we performed transcriptome-wide splicing studies using Arabidopsis thaliana single and double mutants in the NMD factor homologues UPF1 and UPF3, as well as samples treated with the translation inhibitor cycloheximide (CHX). Our analyses revealed that at least 17.5% of all multi-exon, protein-coding genes produce splicing variants of all major types that are targeted by NMD, with a significant fraction originating from the splicing of cryptic introns. Furthermore, accumulation of many intron-retained mRNAs in the mutants, but not in response to CHX suggests the action of distinct routes of NMD with variable impact of translation. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. NMD-dependent AS variants are linked to diverse biological functions, including the poison exon-mediated regulation of signaling and posttranslational protein modification components. In addition to mRNAs, numerous non-coding RNAs as well as newly identified transcripts derived from intergenic regions were shown to be NMD responsive. In summary, our comprehensive analysis of AS-coupled NMD provides strong evidence for a major function of this pathway in shaping the transcriptome, having fundamental implications in gene regulation and quality control of transcript processing steps in higher eukaryotes. Comparison of gene expression and alternative splicing patterns in control and nonsense-mediated decay (NMD)-impaired Arabidopsis seedlings 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 Kahles Drechsel Kahles Kesarwani Stauffer Behr Drewe Rätsch Wachter Person First Name Andre Gabriele Andre Anil Eva Jonas Philipp Gunnar Andreas Person Mid Initials K Person Email akahles@cbio.mskcc.org Person Affiliation Memorial Sloan-Kettering Cancer Center Person Address Computational Biology Center, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, USA Person Roles submitter Protocol Name P-GSE41432-3 P-GSE41432-2 P-GSE41432-1 Protocol Description Illumina Raw Data processing with SHORE 4.1 pipeline with application 'mRNA' and disabled illumina filter Fastq file were aligned to the TAIR10 genome with the spliced aligner PALMapper (version 4) Alignment postprocessing (filtering and multimapper resolution) using RNAgeeq Reads were counted as overlapping to a gene, if they shared at least one exonic position Expression values were transformed to RPKM (Reads per kilobase per million mapped reads) Genome_build: TAIR10 Supplementary_files_format_and_content: Tab delimited text-file, with following columns: (1) gene id; (2) WT; (3) UPF1; (4) UPF3; (5) UPF1UPF3; (6) MOCK; (7) CHX seedlings were ground in liquid N2 and total RNA was isolated using the Universal RNA purification kit (Roboklon) Libraries were prepared using the Illumina TruSeq™ sample prep kit starting from 2-4 ug of total RNA and following the instruction manual (TruSeq RNA Sample Preparation v2 Guide; November 2010) seedlings were grown on 1/2 MS medium containing 2% sucrose for 11 days under continuous light conditions Protocol Type normalization data transformation protocol nucleic acid library construction protocol growth protocol Experimental Factor Name compound dose genotype Experimental Factor Type compound dose genotype Comment[SecondaryAccession] GSE41432 Comment[GEOReleaseDate] 2013-10-01 Comment[ArrayExpressSubmissionDate] 2012-10-09 Comment[GEOLastUpdateDate] 2013-10-01 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE41432_gene_expression_RPKM.txt Comment[SecondaryAccession] SRP016025 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR584115-SRR584126 Comment[AEExperimentDisplayName] Transcription profiling by high throughput sequencing of Arabidopsis upf1, upf3 double mutant and cycloheximide-treated seedlings SDRF File E-GEOD-41432.sdrf.txt