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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.

SUBMITTER: Chapin A

PROVIDER: S-EPMC3962487 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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In vivo determination of direct targets of the nonsense-mediated decay pathway in Drosophila.

Chapin Alex A Hu Hao H Rynearson Shawn G SG Hollien Julie J Yandell Mark M Metzstein Mark M MM

G3 (Bethesda, Md.) 20140320 3

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 o ...[more]

PMID: 24429422

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Project description:Regulation of mRNA stability is a critical step in the control of gene expression. The nonsense mediated mRNA decay (NMD) pathway is one important mRNA stability pathway and has been shown to function in regulating a significant portion of the transcriptome. Perhaps the most significant outstanding question surrounding NMD regulation of endogenous gene expression is the identification of transcripts directly targeted by the NMD pathway versus secondary response genes, whose expression levels are dependent on the activity of direct targets. Here, we present for the first time in an intact animal, the experimental identification of direct targets of NMD. We find that most genes (74%) that are upregulated in NMD mutants appear not to be direct targets of NMD. More surprisingly, we find that the vast majority (97%) of candidate direct targets are not found to be increased in expression in a NMD mutant background. These results imply negative feedback renormalizes the levels of most direct targets of NMD, suggesting they would have been missed in previous analyses. We find that our candidate direct target genes have disproportionately long 3’ UTRs compared to non-targeted control genes. Long 3’ UTRs have been shown to target mRNAs for NMD in experimental systems, and our results imply this is the case in vivo. Our results will allow for the understanding of the function of the NMD pathway in endogenous gene regulation, during normal development, as well as in pathological states. Starting with a Nonsense Mediated Decay(NMD) mutant, in which expression levels of both primary and secondary NMD targets should be increased, we introduce a wild-type copy of the mutated factor and evaluated the transcriptome profile change overtime. Following reintroduction, expression levels of transcripts that are direct targets of NMD should decrease rapidly. Conversely, the levels of indirect targets will remain high until direct targets return to wild-type levels, which will occur later in the timecourse. This allows us to identify direct NMD targets.

Dataset Information

In vivo determination of direct targets of the nonsense-mediated decay pathway in Drosophila.

Publications

In vivo determination of direct targets of the nonsense-mediated decay pathway in Drosophila.

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