PROJECT SUMMARY Protein homeostasis (proteostasis) relies on the continual surveillance and removal of defective translation products resulting from the relatively high error rates associated with mRNA translation. Proteostasis dysfunction has been implicated in human aging-related pathologies, including many neurodegenerative disorders, suggesting that molecular strategies to either limit the production of erroneous translation products or elevate protein quality control capacity may provide therapeutic benefit. As such, characterizing cellular mechanisms that regulate translation activity or ribosome-associated quality control function is needed to enable molecular control over proteostasis under normal and stress conditions. We have discovered conserved, site-specific, regulatory ribosomal ubiquitylation (RRub) events on individual 40S ribosomal proteins that represent a new axis of translational control. Our objective is to determine the molecular mechanisms by which RRub impacts ribosome-associated quality control and the integrated stress response pathway. Toward this goal, we have identified the critical ubiquitin ligases and deubiquitylating enzymes that mediate these RRub events. We have generated a unique and powerful set of genome-edited cell lines that will enable molecular dissection of RRub and the cellular pathways which require RRub for proper function. Our hypothesis is that manipulation of RRub machinery can be utilized to alter translation both during and following acute proteotoxic stress. Furthermore, we hypothesize that cells with elevated translation activity and/or elevated levels of damaged or cleaved mRNAs will require enhanced quality control activity for function and survival. To probe these hypotheses, we will: (1) dissect ubiquitin-dependent and independent mechanisms within the ribosome-associated quality control pathway; (2) determine physiologically-relevant cellular conditions that require elevated RQC activity; and (3) characterize how RRub reshapes translation at steady-state and during activation and recovery of the integrated stress response. Research outcomes achieved by the proposed studies will mechanistically determine how terminally stalled ribosomes are sensed and resolved via the RQC pathway. We will also define how RRub alters stress response pathways through regulation of ribosome abundance or translation activity. Several ribosomal proteins and translation-associated factors are regulatory ubiquitylation targets which suggests that our research strategy can be broadly applied to other targets to enable protein biogenesis control at multiple steps. Successful completion of the proposed research will provide substantial progress toward our long-term goal of combating aging-associated human pathology through the development of molecular strategies to modify cellular responses to chronic proteotoxic stress and improve cellular fitness following proteostasis insults.