Upf1p control of nonsense mRNA translation is regulated by Nmd2p and Upf3p.

The pathways of gene expression include intricate mechanisms that safeguard against the accumulation of aberrant transcripts and proteins (6, 13, 14, 19, 30, 62). In addition to their protective functions, these pathways also contribute additional regulatory facility and complexity (57). The phenomenon of nonsense-mediated mRNA decay (NMD) exemplifies such mechanisms. NMD minimizes the synthesis of truncated polypeptides by eliminating mRNAs containing premature nonsense codons within their protein coding regions (19, 29, 39, 45, 46, 49, 51). NMD also provides the cell with a pathway for the selective degradation of a subset of mRNAs whose coding regions could be considered “normal” (37, 57). In the yeast Saccharomyces cerevisiae, the rapid degradation of nonsense-containing mRNAs proceeds from deadenylation-independent removal of the 5′ cap by the decapping enzyme Dcp1p to 5′→3′ digestion of the remainder of the mRNA by the exoribonuclease Xrn1p (4, 5, 17, 27, 33, 40). Three additional factors are also essential for NMD in yeast: Upf1p, Nmd2p (Upf2p), and Upf3p (7, 20, 22, 34, 35). Mutations in the UPF1, NMD2, or UPF3 genes lead to the stabilization of mRNAs containing premature nonsense codons without affecting the rates of decay of most wild-type mRNAs. Since single or multiple mutations within UPF1, NMD2, or UPF3 yield similar decay phenotypes, all three gene products have been considered to be functionally related and to act in a common pathway (22). Substantial support for this conclusion has been derived from protein-protein interaction analyses (11, 22). A more detailed understanding of the functions of Upf1p, Nmd2p, and Upf3p has been sought in several ways. Consistent with their roles in responding to aberrant translation, all three proteins have been shown to localize to the cytoplasm and to associate with polyribosomes (3, 38, 46). Upf1p is a 109-kDa protein that contains two putative zinc finger domains near its amino terminus and harbors seven motifs characteristic of RNA-DNA helicase superfamily I (1, 31). In vitro studies demonstrated that purified Upf1p has the ability to bind nucleic acids and that its ATPase and helicase activities are dependent upon nucleic acid binding (10, 60). Upf1p interacts with the polypeptide release factors Sup35p and Sup45p (11) and utilizes the same N-terminal zinc finger region for Nmd2p interaction, intramolecular interaction, and homodimerization (F. He and A. Jacobson, unpublished data). Little is known about the biochemical activities of the 127-kDa Nmd2p and 45-kDa Upf3p polypeptides. The involvement of the UPF/NMD genes in regulating the stability of mRNAs containing premature nonsense codons and the interactions of Upf1p with Nmd2p, Upf3p, Sup35p, and Sup45p suggest that UPF1, NMD2, and UPF3 may all be regulators of translation termination and/or fidelity. Consistent with this notion are experiments which indicate that deletion of these genes leads to nonsense suppression (36, 58), allosuppression (9), and enhancement of programmed ribosomal frameshifting (8, 52). To investigate further the possible regulatory roles of Upf1p, Nmd2p, and Upf3p, we devised an assay that quantitatively monitors the effects of upf/nmd mutations on suppression of the can1-100 nonsense allele. Deletion of the genes encoding each of these factors was found to stabilize the can1-100 transcript and promote nonsense suppression. Strains harboring a deletion of UPF1 showed the highest levels of suppression, and overexpression of UPF1 in upf/nmd strains lowered the levels of nonsense suppression significantly without altering the steady-state levels of the can1-100 mRNA. These data and determinations of the abundance of all three factors indicate that Upf1p plays a critical role in regulating the efficiency of translation termination and that Nmd2p and Upf3p, in turn, regulate Upf1p activity.