title: In vivo determination of direct targets of the nonsense-mediated decay pathway in Drosophila.
abstract: Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a mRNA degradation pathway that regulates a significant portion of the transcriptome. The expression levels of numerous genes are known to be altered in NMD mutants, but it is not known which of these transcripts is a direct pathway target. Here, we present the first genome-wide analysis of direct NMD targeting in an intact animal. By using rapid reactivation of the NMD pathway in a Drosophila melanogaster NMD mutant and globally monitoring of changes in mRNA expression levels, we can distinguish between primary and secondary effects of NMD on gene expression. Using this procedure, we identified 168 candidate direct NMD targets in vivo. Remarkably, we found that 81% of direct target genes do not show increased expression levels in an NMD mutant, presumably due to feedback regulation. Because most previous studies have used up-regulation of mRNA expression as the only means to identify NMD-regulated transcripts, our results provide new directions for understanding the roles of the NMD pathway in endogenous gene regulation during animal development and physiology. For instance, we show clearly that direct target genes have longer 3' untranslated regions compared with nontargets, suggesting long 3' untranslated regions target mRNAs for NMD in vivo. In addition, we investigated the role of NMD in suppressing transcriptional noise and found that although the transposable element Copia is up-regulated in NMD mutants, this effect appears to be indirect.
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112.
authors.forename: Alex
authors.initials: A
authors.lastname: Chapin
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.forename: Shawn G
authors.initials: SG
authors.lastname: Rynearson
authors.forename: Julie
authors.initials: J
authors.lastname: Hollien
authors.forename: Mark
authors.initials: M
authors.lastname: Yandell
authors.forename: Mark M
authors.initials: MM
authors.lastname: Metzstein
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D004330:Drosophila / Q000235:genetics* / Q000254:growth & development; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D016678:Genome; D007814:Larva / Q000378:metabolism; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D029721:Drosophila Proteins; D012333:RNA, Messenger; C558301:Upf2 protein, Drosophila
keywords: Drosophila; NMD; RNA-seq; Upf2; reactivation