| Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that recognizes and rapidly degrades mRNAs that undergo premature translation termination. NMD limits expression of truncated and potentially harmful proteins and is essential for survival of mammals. Three Upf proteins in human cells, hUpf1, hUpf2 and hUpf3 form the NMD core, and act in concert with other proteins including hSmg proteins and components of a splicing-dependent exon-junction complex (EJC).; There is contradictory evidence as to whether NMD in human cells is a nuclear or a cytoplasmic process. I show that exogenous expression of interaction domains of a number of NMD factors specifically inhibits NMD. Localization of the inhibitory domains in the nucleus strongly impairs the NMD-inhibitory function, even for those that are designed to inhibit interactions between EJC subunits and hUpf3 proteins, which localize primarily in the nucleus. NMD substrates classified based on cell fractionation assays as "nucleus-associated" (beta-globin and TCR-beta mRNAs) and "cytoplasmic" (GPx1 mRNA) are all inhibited in the same manner. Furthermore, retention of the NMD factor hUpf1 in the nucleus strongly impairs NMD. These observations suggest that the human NMD pathway is primarily, if not entirely, a cytoplasmic process.; The mechanism of premature termination codon (PTC) recognition in mammals is currently believed to depend on downstream introns, and to be mechanistically different from other eukaryotes like yeast where PTCs are defined by "faux" 3'UTRs. Using reporter mRNAs derived from the human beta-globin gene I show that an intron downstream of a termination codon is neither necessary nor sufficient to induce NMD of the mRNA. However, increasing the length of the beta-globin 3' UTR triggers NMD. Tethering PABP1 in proximity of a PTC rescues a PTC-containing beta-globin mRNA from NMD, even when an intron is positioned downstream. These observations suggest that in human cells, like in yeast and Drosophila, termination codons are recognized as PTCs when positioned too far upstream of the polyA-tail due to an aberrant 3'UTR.; Finally, I show that hUpf1, and hSmg proteins involved in its phosphorylation and dephosphorylation, localize to processing bodies (PBs) in human cells. Thus, as in yeast, human NMD pathway also traverses PBs. |
