RNA decay. The balance between RNA transcription and degradation contributes to regulation of RNA lifetime, quality and abundance. Two principle RNA decay pathways exist in eukaryotes, one degrades RNA 5’ to 3’ while the other degrades RNA 3’ to 5’. The 3’ to 5’ decay pathway requires activities of the RNA exosome, a multi-subunit protein complex that contains a nine-subunit non-catalytic core and two additional subunits that catalyze processive and distributive 3’ to 5’ RNA exoribonuclease activities. Ten of the eleven genes are essential for life in budding yeast, suggesting the importance of the RNA exosome and its activities in cellular function. In addition, reports over the last several years suggest that humans harboring mutations in select components of the 3’ to 5’ decay pathway suffer from diseases ranging from motor neuronopathies to cancer. Fundamental aspects of eukaryotic exosome structure and function have been illuminated; however, many questions remain with respect to how upstream factors target substrates for degradation. As RNA decay pathways play a fundamental role in eukaryotic nucleic acid metabolism and disease, our studies are directly relevant to human health and the NIH mission as misregulation of RNA decay is associated with cancer, inflammation and neurodegeneration. This renewal will address central issues of RNA exosome biology by reconstituting or purifying RNA exosomes and upstream factors for characterization through biochemical, genetic and structural studies to establish functions for 3’ to 5’ decay in vitro and in vivo. Ubiquitin-like proteins. Signal transduction can rely on reversible chemical modifications to relay information. Protein substrates can be covalently modified by ubiquitin and ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) to regulate processes such as nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcription. Post-translational modification by ubiquitin (Ub) and ubiquitin- like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 protein ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play integral roles in eukaryotic nuclear metabolism and cell cycle control and our studies are of direct relevance to human health, cancer, and the mission of the NIH. This renewal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish a basis for Ub/Ubl activation, conjugation by E2 and E3 enzymes, and signaling through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and cofactors utilized for ubiquitin and SUMO protein conjugation pathways are conserved so our studies are broadly relevant to other Ub/Ubl-related pathways.