Degradation of mRNA plays an important role in regulating gene expression. In eukaryotic cells a specialized pathway has evolved to stimulate the degradation of aberrant mRNAs containing a premature nonsense codon (PTC), a signal that causes early termination of translation and, if left unchecked, the accumulation of truncated polypeptides. This pathway, referred to as nonsense- mediated mRNA decay (NMD), represents a conserved quality control mechanism that upholds fidelity and protects cells from erroneous gene expression. The widely accepted model for NMD posits that PTC-containing mRNA are recognized as aberrant due an impaired interaction between the terminating ribosome and the mRNA poly(A) tail-bound protein, PAB1, as a consequence of translation termination occurring too far away. Our observations challenge this paradigm and indicate that a model invoking a distance between the terminating ribosome and PAB1 as the critical determinant in NMD substrate recognition cannot sufficiently explain how cells discriminate between normal and premature translation termination. The long term goal of this proposal is to elucidate the molecular mechanisms underlying both recognition and rapid degradation of mRNA by the NMD pathway. We will address the important questions of how nonsense-containing mRNA are both recognized and targeted for rapid degradation under three specific aims. First, we will identify and characterize mRNP elements important for targeting PTC-containing mRNA to NMD. In addition, we propose two innovative and unbiased approaches to identify cis- and trans-acting antagonists of NMD. Second, we will investigate the requirements for assembly of NMD proteins with mRNA substrates. In particular, we will examine the order for protein assembly, whether binding targets are ribosomes, mRNA, or both, and particular mRNA features for their importance in NMD mRNP assembly. Finally we will also assess the functional capabilities of individual NMD proteins independent of their RNA binding capacities. Third, we will elucidate how recognition of a PTC- containing mRNA as aberrant is communicated to the cellular decay machinery and leads to accelerated degradation of the mRNA. Our preliminary data dispute a common belief that the NMD machinery directly recruits the decay machinery to the mRNA through conserved protein- protein interactions. We plan to further challenge this model as well as test our hypothesis that rapid mRNA decay is an indirect consequence of restricting translation initiation factor binding to the mRNA 52 cap. PUBLIC HEALTH RELEVANCE: Cells possess elaborate mechanisms to ensure that the expression of genetic information is accurate and to safeguard against the accumulation of aberrant mRNA and proteins. The pathway described in this grant, nonsense-mediated mRNA decay (NMD), represents a conserved quality control response in cells that recognizes and eliminates mRNA with a specific class of mutation common to one-third of all human genetic diseases. The implications of this proposal on human health are high since a detailed understanding of NMD will undoubtedly lead to novel therapeutic approaches to prevent suffering in patients with this class of genetic mutation.