Most non-sense mRNAs are highly unstable because they are degraded by a decay pathway called nonsense-mediated mRNA decay (NMD). The process whereby mRNAs are monitored to eliminate those that code for potentially deleterious protein fragments is called RNA surveillance. Surveillance occurs in fungi, plants, nematodes, and vertebrates including humans. The proteins that catalyze steps in NMD, called Upf proteins, serve two roles, one to monitor errors in gene expression and the other to control the abundance of endogenous wild-type mRNAs as part of the normal repertoire of gene expression. Studies of the proteins required for NMD hold great promise for telling us home errors in gene expression are monitored and how gene expression is controlled by noel post-transcriptional mechanisms. The NMD pathway has a direct impact on hundreds of genetic disorders in the human population, where one fourth to one third of all mutations causing inherited genetic disorders and various forms of cancer are of the type that could target the corresponding mRNA for NMD. The primary thrust of this proposal is to understand the mechanism of NMD, the connection between NMD and translation, and the role of NMD in RNA surveillance and gene expression. The yeast Saccharomyces cerevisiae will be exploited as a model system applicable to complex eukaryotes. We will establish the role of Upf2 and Upf3 in the recruitment of nonsense mRNAs into the NMD pathway. The hypothesis we have put forward is that recruitment requir5es the shuttling Upf3p and possibly Upf2p between the nucleus and cytoplasm to mark nonsense mRNAs in the nucleus for subsequent decay in the cytoplasm. We will examine Upf-like suppressors in two new genes that have been identified. One of these suppressor mutations residues in the EGS1 gene, which is of interest because of its potential role in translation and because its expression is controlled by the NMD pathway. Our hypothesis for the mechanism of NMD is that Upf3p acts first by binding to mRNP particles in the nucleus after which it exports to the cytoplasm as an mRNP-bound protein. We will address various aspects of this model by using tethering assays, genetic studies of mRNP export, and biochemical analysis of mRNP's. Finally, we will examine the role of Upf proteins in global gene expression. Using DNA arrays, we have identified over 500 genes whose expression is affected by the Upf proteins. We will identify targets and distinguish direct from indirect targets of the UPF genes, establish mechanisms of targeting, and characterize the global gene expression profile for uPF-strains.
