PROJECT SUMMARY Post-transcriptional mechanisms play a fundamental role in regulating gene expression at the protein level, and are frequently implicated in stress response, aging, and diseases. The goal of this project is to develop and apply multi-omics methods to examine the post-transcriptional mechanisms that regulate protein composition of multiple tissues and their ability to respond to proteostatic stressors. In recent work, our team has developed mass spectrometry and multi-omics methods that are designed to elucidate the protein isoform composition and spatiotemporal dynamics. Building on these progresses, we will focus here on the roles of three post-transcriptional mechanisms known to influence protein translation in stress response. Specifically, Aim 1 will integrate proteomics and transcriptomics data to identify the role of alternative splicing in modulating principal isoform abundance, creating alternative proteoforms, and influencing protein localization in mammalian tissues. Aim 2 will determine the differential expression, localization, and targets of RNA-binding proteins in proteostatic stress responses including paraquat in vivo as well as doxorubicin and hydrogen peroxide in vitro. Finally, Aim 3 will examine the configuration and interactome of the translation apparatus including the core ribosome and an increasing number of known ribosome-associated proteins, which have emerged as important factors that can fine-tune the translational efficiency of individual transcripts and the associated protein synthesis rates. The proposed experiments will interrogate the relationships between post-transcriptional regulation and stress response, and at the same time generate novel data sets including isoform-resolved, spatiotemporal atlases of the normal, stressed, and aged/senescent proteomes. We anticipate the results will lead to novel insights into basic cellular processes of stress response and resilience that will be relevant to studies of multiple systems.