hUPF2 Silencing Identifies Physiologic Substrates of Mammalian Nonsense-Mediated mRNA Decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that selectively degrades aberrant mRNAs with premature termination codons (PTCs). Although a small number of cases exist in mammals, where NMD controls levels of physiologic PTC transcripts, it is still unclear whether the engagement of NMD in posttranscriptional control of gene expression is a more prevalent phenomenon. To identify physiologic NMD substrates and to study how NMD silencing affects the overall dynamics of a cell, we stably down-regulated hUPF2, the human homolog of the yeast NMD factor UPF2, by RNA interference. As expected, hUPF2-silenced HeLa cells were impaired in their ability to recognize ectopically expressed aberrant PTC transcripts. Surprisingly, hUPF2 silencing did not affect cell growth and viability but clearly diminished phosphorylation of hUPF1, suggesting a role of hUPF2 in modulating NMD activity through phosphorylation of hUPF1. Genome-wide DNA microarray expression profiling identified 37 novel up-regulated and 57 down-regulated transcripts in hUPF2-silenced cells. About 60% of the up-regulated mRNAs carry typical NMD motifs. Hence, NMD is important not only for maintaining the transcriptome integrity by removing nonfunctional and aberrant PTC-bearing transcripts but also for posttranscriptional control of selected physiologic transcripts with NMD features.

Supplemental material for this article may be found at http://mcb.asm.org/.

Part of this work was supported by the Human Frontier Science Program, the Deutsche Forschungsgemeinschaft (DFG), and the Hertha Löw Foundation to H.-M. Jäck.

We thank Reuven Agami for providing pSUPER; Andreas Kulozik for β-globin WT and nonsense (PTC) constructs; Joachim Lingner and Claus Azzalin for pSUPER-E8 and -E10; Ludger Klein-Hitpass for performing DNA microarray experiments; Brett Lane for help in preparing hUPF2 antibodies; Anja Haude and Edith Roth for technical assistance; and Harald Bradl, Dirk Mielenz, Johannes Lutz, and Christian Vettermann for comments on the manuscript.