Nonsense-Containing mRNAs That Accumulate in the Absence of a Functional Nonsense-Mediated mRNA Decay Pathway Are Destabilized Rapidly upon Its Restitution

Abstract

Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins. Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm. Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway. To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter. The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced. Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min. These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation.

ACKNOWLEDGMENTS

This work was supported by a grant (GM27757) to A.J. from the National Institutes of Health and a predoctoral NRSA fellowship (GM18043) to A.M. from the National Institutes of Health.

We thank the members of the Jacobson laboratory for their helpful advice and editorial comments and are especially indebted to David Mangus for stimulating insights.

Alan B. Maderazo and Jonathan P. Belk contributed equally to this work.