(A) Gadd45 mRNA expression in adults of the given genotypes measured by qRT-PCR. Gadd45 mRNA expression is increased 16.7-fold in Upf225G mutants, and is eliminated by Gadd45F17 mutants. p-values display one-sided Student’s t-test of indicated condition compared to control. Error bars represent 2 SEM. (B) Fluorescence of GFP transgenes with SV40, Act5C, or Gadd45 3’ UTRs expressed by UAS driven by Actin:GAL4 in Upf2+ or Upf225G third instar larvae. SV40 and Gadd45 3’ UTR constructs show significantly increased fluorescence in Upf225G animals compared to Upf2+, indicating NMD-dependent post-transcriptional degradation of mRNAs containing these UTRs. The Act5C 3’ UTR construct has similar fluorescence in both backgrounds, indicating NMD does not regulate the post-transcriptional stability of this UTR. Micrographs show dorsal views with anterior at top. (C) MAL-A2, traL, and Gadd45 5’ and 3’ fragment mRNA expression measured by qRT-PCR in pcm14 null mutants (Waldron et al., 2015) normalized to controls. Transcript structures of a non-NMD-target, maltase A2 (MAL-A2); a known NMD-targeted transcript, the non-sex specific isoform of transformer (traL) (Rehwinkel, 2005; Metzstein and Krasnow, 2006); and the Gadd45 transcript (note Gadd45 has no introns). Open boxes indicate UTRs; grey boxes indicate coding regions. NMD targeting initiates endonucleolytic cleavage near the stop codon (Gatfield and Izaurralde, 2004), producing 5’ and 3’ fragments with unprotected ends, which are then subjected to degradation by cytoplasmic 3’-to-5’ and 5’-to-3’ exonucleases, respectively. qRT-PCR primer pairs 5’ (red) and 3’ (blue) to the cleavage site can be used to differentially measure the quantity of these fragments. The Drosophila 5’-to-3’ exonuclease is encoded by the XRN1 homologue pacman (pcm), and fragments 3’ to an endonucleolytic NMD cleavage accumulate in Drosophila cells with reduced XRN1 activity (Gatfield and Izaurralde, 2004). The MAL-A2 3’ primers show no difference in relative expression in pcm14 mutants compared to the 5’ primers, while tra and Gadd45 have relatively increased levels of a 3’ fragment in pcm14 mutants, revealing endonucleolytic cleavage has occurred between the primer pairs, probably near the stop codon, indicative of NMD-initiated degradation. p-value between indicated samples using a two-sided Student’s t-test are displayed. ns indicates a p-value greater than 0.05. Error bars represent 2 SEM.