Post-transcriptional regulations of proteomes in stress and senescence

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Project Summary This administrative supplement application is being submitted to PA-20-272 in accordance with NOT-GM-22- 017. The scope of the parent project R01-GM144456 will not be changed. The requested supplement will replace critical hardware infrastructure to accelerate our research with modern capabilities and permit increased productivity. Parent 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 intrinsic and environmental 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 across tissues (heart, lung, muscle, brain), sexes, and lifespan. Aim 2 will determine the differential expression, localization, and targets of RNA-binding proteins in proteostatic stress responses to intrinsic or extrinsic stressors 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.