Project Summary/Abstract Mechanistically understanding how cells rapidly respond to and survive environmental stresses is crucial for gaining insight into treatment and prevention of human diseases – many of which are stress manifest in different forms. Decreasing cellular energy requirements while simultaneously rapidly, yet precisely, regulating gene expression is a particular challenge for cells under stress. Dynamic subcellular compartments that respond to stress may contribute to the cell’s integrated stress response. Stress granules (SGs) and processing bodies (PBs) are both stress-responsive temporary cytoplasmic compartments comprised of RNA and proteins. Inhibition of protein synthesis frees messenger RNA (mRNA) from the translation machinery, providing substrate for Liquid-Liquid Phase Separation (LLPS), which gives rise to SGs. PBs, while often constitutively present, respond to the shut-down of translation by increasing in size and number. While SGs have received much attention and several lines of evidence suggest they contribute to cell survival under stress, less is understood about PBs. A few recent studies have converged on a new protein, Ubiquitin Associated Protein 2-like (UBAP2L) as a critical component of SG assembly via LLPS. Interestingly, we have recently discovered that not only does UBAP2L localize to SGs and contribute to their formation, but it appears to regulate physical linkage of SGs and PBs. Typically, stress causes the formation of SGs with PBs attached. Our preliminary data show that in the absence of UBAP2L, formation of SGs with attached PBs is impaired, while overexpression of UBAP2L results in mixed granules consisting of proteins typically found only either in SGs or PBs. Additionally, we found that knockout of UBAP2L reduces basal translation levels, suggesting that UBAP2L may modulate translation. This hypothesis has been confirmed by others who demonstrate UBAP2L enhances translation of essential protein synthesis genes. We propose to 1. Determine the mechanism by which UBAP2L contributes to the formation of SGs with attached PBs, upon stress; 2. Determine the mechanism by which UBAP2L specifically modulates translation of TOP-mRNAs (a class of mRNAs essential to protein synthesis) and how this modulation changes under stress. Additionally, our synergistic aims will allow us to determine if there is a connection between UBAP2L’s role in RNA granule interaction and translation regulation, or if different regions of the protein contribute to these distinct roles. This proposal investigates novel roles of UBAP2L and promises important contributions to understanding the stress response and protein biology. Successful completion of this proposal will yield a mechanistic understanding of UBAP2L’s contribution to the stress response and therefore may be important in advancing therapeutics, particularly for treatment of diseases characterized by abnormal RNA granule assembly or disassembly.