PROJECT SUMMARY/ABSTRACT Gene expression is closely monitored to ensure fidelity in the conversion of genetic information into biological activity. In eukaryotic cells a specialized quality control checkpoint exists during mRNA translation to evaluate mRNA integrity and rapidly degrade transcripts containing a nonsense codon in their protein-coding region - a signal that causes premature termination of translation and, if left unchecked, the accumulation of truncated polypeptides. This pathway, referred to as nonsense-mediated mRNA decay (NMD), serves a vital biological function by protecting cells from the deleterious consequences of incomplete proteins. In spite of over two decades of study, fundamental questions central to this process remain poorly resolved, including how cells distinguish normal and premature translation termination and what molecular events occur subsequently between the translating mRNA and NMD machinery to incite accelerated degradation of the nonsense codon- containing transcript. The long term objective of my research is to gain a comprehensive molecular understanding of the events underlying the cell's ability to recognize and target nonsense-containing mRNA for rapid decay. In the last decade, we have made seminal discoveries in characterizing RNA features vital for the recognition and efficient targeting of an mRNA to NMD. Recently, we have begun to dissect molecular events subsequent to substrate recognition through identifying the complement of proteins specifically associated with nonsense- containing mRNA and characterizing a catalytically-inactive mutant of the key NMD factor, UFP1. Notably, we have described a functional interaction between the NMD machinery and prematurely terminating ribosomes that exists in vivo and which is critical for dictating the fate of the aberrant transcript. We propose here to capitalize on our past efforts and exploit novel tools and reagents to i) identify UPF1 RNA binding targets globally to provide unprecedented insight into how NMD factor binding dictates the targeting of mRNA to NMD; ii) interrogate further the functional interaction between prematurely terminating ribosomes and the NMD machinery essential for targeting substrates to rapid decay; and iii) monitor NMD mRNP dynamics in vivo using enzyme-catalyzed proximity labeling. Together, these studies will provide unprecedented insight into the molecular events underlying an integral cellular RNA quality control pathway and regulator of gene expression.