Project Summary This project pursues two research directions that build on two NIGMS funded research programs. Both projects are loosely connected and the pathways studied will help understand how metabolic and environmental cues are sensed and transmitted to the cell cycle machinery. Previous work in yeast models has established different ubiquitin-mediated signaling events that communicate metabolic and environmental states to the cell cycle machinery. Molecular understanding of the concepts that govern ubiquitin signaling are thus the topic of the first part of this application. Project-1 studies both yeast and mammalian cell line models to define general concepts of ubiquitin signaling. These experiments build on a plethora of tools we have developed to analyze biochemistry and physiology of ubiquitin signaling and will address the following questions: (1) How do readers of the ubiquitin signal distinguish different chain types? (2) How do F-box proteins sense metabolic and environmental states? (3) How do ubiquitin ligases recognize substrates in a context specific manner? (4) How is signaling achieved by phosphorylated ubiquitin. Proposed work in project 1 will define detailed molecular insight in aspects of ubiquitin signaling both proteolytic and non-proteolytic. Project-2 is focused on the mammalian system, were we discovered important connectors between methionine metabolism and cell proliferation. These include regulation of protein phosphatase 2A (PP2A) and RNA CAP methylation of selected transcripts. Work on PP2A in mammalian cells will focus on the role of methylation of the carboxy terminus of the catalytic subunit of PP2A as a sensor of methionine metabolism. Experiments will expand on proteomic profiling that identified several PP2A interaction partners with preference for the demethylated PP2A complex. We will dissect these interactions and define their role in communicating metabolic states to the cell cycle machinery. Studies related to mRNA CAP methylation will extend our recent findings that a small group of RNAs is highly sensitive to subtle fluctuations in the cellular methylation potential, which is controlled by methionine metabolism. These RNAs become hypomethylated on their mRNA CAPs, and thus are inefficiently translated when methionine is limiting. The goal is to understand mechanisms that make certain mRNAs hypersensitive to fluctuation in methionine metabolism, and to discover the mechanistic link to cell cycle control. Understanding the molecular concepts that integrate methionine metabolism with cell proliferation promise new therapeutic strategies, especially for the treatment of cancer and other age-related disorders. Thus, this proposal aims to development molecular insight into a fundamental, so far molecularly unexplored, biological process with great therapeutic potential.