The nonsense-mediated mRNA decay (NMD) pathway of the yeast Saccharomyces cerevisiae is an example of a specialized mRNA decay pathway. Targets of this pathway include mRNAs that have premature translation termination codons (nonsense mRNAs), and several wild-type RNAs. The process by which wild-type mRNAs are recognized and targeted for accelerated decay by the NMD pathway is presently unknown, and the cellular compartment in which NMD occurs is controversial. To begin to understand how wild-type mRNAs are recognized and targeted for NMD, it will be essential to characterize wild-type targets of the NMD pathway. PPR1 mRNA is the first identified wild-type mRNA targeted for accelerated mRNA decay by the NMD pathway. The cis-acting element(s) within the PPR1 mRNA that target this mRNA for accelerated decay by the NMD pathway will be identified by a combination of fusion constructs, deletions and point mutations. If the PPR1 cis-acting sequences are linear, as opposed to defining a characteristic secondary structure, these sequences will then be used to screen for other potential wild-type targets of the NMD pathway. Translation appears to be required for NMD, suggesting that NMD occurs in the cytoplasm. Consistent with that idea, Upf1p, Upf2p and Upf3p, three proteins required for NMD, are associated with ribosomes. However, studies in mammals have implicated a role for the nucleus in NMD. Upf1p interacts with Nup100p, a component of the nuclear pore complex that has a role in mRNA transport. The biological significance of the interaction between these proteins will be determined using co-immunoprecipitation and genetic approaches.
mRNA degradation is an important step for regulating gene expression. The mechanism by which mRNAs are targeted for decay by specialized mRNA decay pathways is beginning to be understood. This project will study a particular decay pathway, called nonsense-mediated decay (NMD), which is responsible for the degradation of mutant mRNAs and at least one wild-type mRNA. The sequences in the wild-type mRNA that target it for decay by the NMD pathway will be determined. Furthermore, proteins that interact with the NMD pathway and components of the nuclear envelope will be characterized. These results will be significant because they may begin to explain the observation, made in mammalian studies, that the nucleus plays a role in NMD. Since NMD is conserved in organisms ranging from Escherichia coli to humans, studies on NMD in yeast will have relevance to a broad range of organisms.
