Identification of directly targeted mRNA substrates of the NMD pathway. Alex Chapin, Mark Metzstein. Human Genetics, University of Utah, Salt Lake City, UT.

   Nonsense mediated mRNA decay (NMD) is an evolutionary conserved post-transcriptional regulatory pathway which, in addition to targeting mRNAs harboring premature termination codons for rapid degradation, is also necessary for normal gene expression. Mutations in NMD genes result in upregulation of numerous endogenous transcripts and in Drosophila, like other complex organisms, NMD genes are vital, likely due to misregulation of certain endogenous targets. However, the identity of the critical target genes is not known. Complicating the discovery of critical NMD targets, is that it cannot be easily distinguished which of the overexpressed genes are directly regulated by the NMD machinery, from those whose expression changes in response to overexpression of direct targets. To address this, we have developed a novel, kinetics-based assay to identify direct targets of NMD in whole animals. In our assay, wild-type Upf2 (a core component of the NMD machinery) is resupplied to Upf2 mutant larvae using a heat inducible transgene. The transcriptome of these animals is then monitored over time using microarray analysis. In this experiment, it is expected that levels of direct targets will quickly return to baseline, while levels of indirect targets are restored more slowly. Using this approach, we have identified a candidate set of direct targets. One of these is the evolutionary conserved gene Gadd45. Gadd45 is activated in response to various cellular stresses to mediate growth arrest, apoptosis and transcription of stress related genes. Interestingly, defects found in animals overexpressing only Gadd45 mirror those of NMD loss-of-function mutants, including cell cycle, and embryonic patterning. We have also found that genomic deficiencies which remove Gadd45, or the immediate downstream transducer of Gadd45 activity, Mekk1, can suppress lethality in Upf2 mutants. Our results suggest that Gadd45 is one of, if not the, critical downstream effector of NMD-mediated lethality.