Rapid Deadenylation Triggered by a Nonsense Codon Precedes Decay of the RNA Body in a Mammalian Cytoplasmic Nonsense-Mediated Decay Pathway

Abstract

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5′-to-3′ exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.

ACKNOWLEDGMENTS

We thank T.-C. Chang, A. van Hoof, and R. Kulmacz for critical reading of the manuscript and for their valuable comments and L. Maquat for providing us with expression plasmids containing Flag-tagged hUpf1 wild-type cDNA and R844C mutant cDNA. Special thanks go to S. Durdan, N. Xu, and W. Zhu for technical assistance.

This work was supported by grants from the National Institutes of Health to A.-B. S. (GM59211 and GM46465).