The nonsense-mediated mRNA decay (NMD) pathway monitors premature translation termination and degrades aberrant mRNAs. In yeast, it has been proposed that a surveillance complex searches 3' of a nonsense codon for a downstream sequence element (DSE) associated with RNA-binding proteins. An interaction between the complex and the DSE-binding protein(s) triggers NMD. Here we describe the identification and characterization of the Hrp1/Nab4 protein as a DSE-binding factor that activates NMD. Mutations in HRP1 stabilize nonsense-containing transcripts without affecting the decay of wild-type mRNAs. Hrp1p binds specifically to a DSE-containing RNA and interacts with Upf1p, a component of the surveillance complex. A mutation in HRP1 that stabilizes nonsense-containing mRNAs abolishes its affinity for the DSE and fails to interact with Upf1p. We present a model describing how Hrp1p marks a transcript for rapid decay.
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysomeassociated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.
Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.Cells have evolved many quality control mechanisms to eliminate aberrant proteins and mRNAs that interfere with normal cellular functions. One such mechanism is the nonsense-mediated mRNA decay (NMD) pathway, which eliminates mRNAs that contain premature termination (nonsense) codons within the protein coding region, thereby preventing the synthesis of truncated proteins with dominant-negative and deleterious gainof-function activities (3,7,14,16,24,42,47). The importance of this surveillance mechanism is underscored by its conservation in a wide variety of organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, plants, and mammals (12,13,16,25,27,34,38).Several genes essential for NMD have been identified in yeast, most notably UPF1, UPF2, and UPF3, all of which destabilize nonsense codon-containing mRNAs without affecting the decay rate of most wild-type mRNAs (16,25). The yeast UPF1 gene encodes the protein Upf1p, which has RNA-binding and RNA-dependent ATPase/helicase activities (8, 9, 45). Yeast UPF3 encodes the basic protein Upf3p, which harbors several nuclear localization and nuclear export signals that allow the protein to shuttle between the nucleus and the cytoplasm (39, 40). Yeast UPF2 encodes the adaptor protein Upf2p, which forms a complex with both Upf1p and Upf3p (6, 19). Single or multiple deletions of each of these three UPF genes produce similar effects on mRNA decay, consistent with the notion that the Upf proteins function as a molecular complex in a single pathway (19).The NMD pathway is elicited by recognition of a nonsense codon, but the precise features distinguishing a premature termination codon from a bona fide stop codon remain unknown. In S. cerevisiae, one model for NMD suggests that the de...
Eukaryotes have evolved conserved mechanisms to rid cells of faulty gene products that can interfere with cell function. mRNA surveillance is an example of a pathway that monitors the translation termination process and promotes degradation of transcripts harboring premature translation termination codons. Studies on the mechanism of mRNA surveillance in yeast and humans suggest a common mechanism where a “surveillance complex” monitors the translation process and determines whether translation termination has occurred at the correct position within the mRNA. A model will be presented that suggests that the surveillance complex assesses translation termination by monitoring the transition of an RNP as it is converted from a nuclear to a cytoplasmic form during the initial rounds of translation. BioEssays 21:685–696, 1999. © 1999 John Wiley & Sons, Inc.
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