Abstract One third of inherited human genetic diseases are caused by mRNAs harboring premature termination codons (PTCs) as a result of nonsense or frameshift mutations. These types of mutations give rise to aberrant transcripts that are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. This RNA surveillance pathway is important, as it greatly reduces the synthesis of truncated proteins, some of which possess deleterious gain-of-function or dominant-negative effects. In addition, recently it has become clear that NMD regulates transcripts from about 5% of normal genes. This suggests that NMD is not only an RNA surveillance pathway but also performs a regulatory role in gene expression. The core genes involved in NMD (UPF1, UPF2, and LJPF3) were first identified in Saccharomyces cerevisiae. Orthologues of these genes have also been identified in Caenorhabditis elegans, Drosophila melanogaster, and humans, suggesting that NMD is a highly conserved RNA surveillance mechanism in eukaryotes. While there is increasing evidence for the physiological relevance of NMD, the underlying mechanism and regulation of this RNA surveillance pathway remains poorly understood. A particularly important issue that has only begun to be addressed is the role of phosphorylation in NMD. Numerous studies have provided evidence that both the phosphorylation and dephosphorylation of the RNA helicase UPF1 have a role in NMD in many organisms. However, the functional residues phosphorylated in UPF1 and the biochemical relevance of UPF1 phosphorylation and dephosphorylation has not been clearly elucidated in any organism. Likewise, UPF2 has been shown to be phosphorylated but it is not known why. This proposal fills this gap by identifying and functionally testing the conserved sites of UPF1 and UPF2 phosphorylation using a combination of molecular, biochemical, and genetic approaches in Saccharomyces cerevisiae and mammalian cell lines. A better understanding of the NMD pathway may permit the discovery of approaches to modulate the stability and translation of aberrant mRNAs as a means to combat cancer and other human genetic disorders caused by nonsense and frameshift mutations.