In vivo determination of candidate direct targets of the nonsense mediated decay pathway in intact Drosophila

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Hao Hu 

PROVIDER: E-GEOD-47979 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress