In VivoDetermination of Direct Targets of the Nonsense-Mediated Decay Pathway inDrosophila

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.

Stress and the nonsense-mediated RNA decay pathway

“…While it is not known how uORFs trigger NMD, it may be through EJCs just downstream of the uORF [10, 16, 70]. A third NMD-inducing feature is long 3′UTRs, which may act by increasing the distance of factors bound at the site of translation termination and those bound at the poly(A) tail [7, 14, 19, 71, 72]. …”

“…They found that Gadd45 / Upf2 double-mutant flies had significantly larger eye patch size than Upf2 -mutant flies, providing further evidence that loss of Gadd45 improves the viability of NMD-deficient cells [74]. These findings, coupled with the fact that GADD45 is a pro-apoptotic molecule [126, 127], and it is encoded by a mRNA that is a direct NMD target transcript [71], led Nelson et al to propose a model in which NMD normally degrades Gadd45 transcripts to avoid its pro-apoptotic effects. In scenarios in which NMD is suppressed, such as stress, Gadd45 transcripts are stabilized, leading to increased GADD45 protein expression and consequent apoptosis [74].…”

The Y Chromosome Modulates Splicing and Sex-Biased Intron Retention Rates in Drosophila

“…For instance, a fraction of the sex-biasedly retained introns do not contrain a PTC and could be competent to produce functional proteins. It is also noteworthy that many PTC containing mRNAs are not affected by the NMD process (HANSEN et al 2009;SAULIERE et al 2010;CHAPIN et al 2014), suggesting the existence of PTC containing IR transcripts that are not subject to NMD. We also observed a mild enrichment for sex-biased IR events in UTR regions ( Figure 2C) compared to the overall distribution.…”

Unique Aspects of Plant Nonsense-Mediated mRNA Decay

“…This obstructs the reliable prediction of NMD-targeted transcripts on the basis of EEJ position information. Direct NMD targets were determined by several strategies in yeast, Drosophila, and mammalian cells [32,105,106], but their identification in plants is still awaited.…”