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

Upf1p, Nmd2p, and Upf3p regulate the degradation of yeast mRNAs that contai n premature translation termination codons. These proteins also appear to r egulate the fidelity of termination, allowing translational suppression in their absence. Here, we have devised a novel quantitative assay for transla tional suppression, based on a nonsense allele of the CAN1 gene (can1-100), and used it to determine the regulatory roles of the UPF/NMD gene products . Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and pr omoted can1-100 nonsense suppression. Changes in mRNA levels were not the b asis of suppression, however, since deletion of DCP1 or XRN1 or high-copy-n umber can1-100 expression in wild-type cells caused an increase in mRNA abu ndance similar to that obtained in upf/nmd cells but did not result in comp arable suppression. can1-100 suppression was highest in cells harboring a d eletion of UPF1, and overexpression of UPF1 in cells with individual or mul tiple upf/nmd mutations lowered the level of nonsense suppression without a ffecting the abundance of the can1-100 mRNA. Our findings indicate that Nmd 2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role i n promoting termination fidelity that is independent of its role in regulat ing mRNA decay. Consistent with these relationships, Upf1p, Nmd2p, and Upf3 p were shown to be present at 1,600, 160, and 80 molecules per cell, levels that underscored the importance of Upf1p but minimized the likelihood that these proteins were associated with all ribosomes or that they functioned as a stoichiometric complex.