Impact of Nonsense-Mediated mRNA Decay on the Global Expression Profile of Budding Yeast

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd+) and mutant (Nmd−) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% ± 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd− strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Association of yeast Upf1p with direct substrates of the NMD pathway

“…The percentage of direct substrates estimated here is comparable with that obtained in the genomewide mRNA half-life experiment, where 278 of 598 NMD-regulated transcripts were proposed to be direct substrates (11). However, there may be significant difficulty in accurately predicting substrates from global decay rate experiments, a conclusion evident from the lack of correlation between the direct and indirect substrates identified by Guan et al (11) and either the Upf1p-associated mRNAs or the mRNAs defined herein as direct substrates (SI Fig.…”

“…However, the basis for this modulation of mRNA levels has been unclear, i.e., it is unknown whether most NMD-regulated transcripts are directly targeted for accelerated degradation by the NMD pathway, or if their levels change as an indirect consequence of the altered expression of a relatively small number of genuine substrates. Three genomewide approaches have been used to address this problem, namely the determination of mRNA half-lives in NMD-deficient cells (11), the identification of mRNAs whose abundance decreases when NMD is reactivated (Fig. 1), and the characterization of mRNAs that copurify with Upf1p (Figs.…”

“…Previous studies have suggested that the NMD pathway may be involved in the regulation of specific cellular processes (3,5,7,11). An examination of the gene ontology classifications associated with the NMD-regulated yeast transcripts revealed that the process with the highest likelihood of being NMD-regulated was thiamine metabolism.…”

“…Further analyses of the transcripts up-regulated when NMD is inactivated suggested that NMD not only controls the expression of aberrant transcripts, but also that of many apparently wildtype mRNAs. This finding, in combination with the observation that the transcripts displayed an overrepresentation in specific cellular processes, has suggested that NMD may have a regulatory function beyond mRNA surveillance (3,5,(7)(8)(9)11). However, comparisons of NMD-regulated transcripts and processes between different organisms have revealed a poor overlap, suggesting that the regulatory role is not evolutionarily conserved (7,9).…”

“…This view is supported by the observation that none of nine NMD-regulated transcripts defined in another study showed altered half-lives in a strain lacking NMD (4). More recently, genomewide mRNA half-life determinations indicated that Ϸ50% of the transcripts that accumulate in NMD-deficient cells are direct NMD substrates (11). However, the intrinsic difficulties in such half-life determinations suggested that it was important to use an independent approach to classify the substrates.…”