Candidate’s Project Abstract: Biological phase separation phenomena contribute to many fundamental cellular organization and regulatory processes, from transcription to post-transcriptional gene regulation, and have recently been identified as a pathophysiological hallmark of two prominent neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Stress granules (SGs), macromolecular complexes of RNAs and RNA binding proteins (RBPs) that assemble in response to stress-elicited inactivation of translation initiation, are an actively studied example of a regulatory biological phase separation process and a prominent experimental model in the field. Despite the well-established finding that SG biogenesis is a ubiquitous cellular response to stress-induced translational suppression, there remain substantial gaps in our understanding of SG biogenesis. Among the most significant gaps is an understanding of the biological criteria for mRNA recruitment into SGs. Transcriptome analyses of isolated SGs have provided limited insights into SG recruitment criteria, revealing a positive correlation with CDS and UTR length and a negative correlation with translational efficiencies. An additional and unexpected finding from SG transcriptome analyses was that endoplasmic reticulum (ER)- targeted mRNAs are substantially under-represented in SG mRNA transcriptomes. This finding is all the more unexpected in light of recent finding that ER dynamics are functionally coupled to SG biogenesis and regulate granule biogenesis and fusion. How the ER contributes to SG biogenesis while its associated mRNAs are largely sequestered from these regulatory organelles is unknown. An additional important and as yet unexplained observation is that on average, only a small fraction (< 10%) of a given mRNA is recruited into SGs. It is not currently known if this fraction comprises a random, stochastic sampling of the cellular mRNA population or, alternatively, a distinguishable cohort of mRNAs. Addressing these gaps in knowledge will be accelerated by experimental models that enable molecular dissection of these questions. To this end, we recently discovered that SGs assemble on the ER in response to activation of the unfolded protein response (UPR) and, critically, that mRNA recruitment into ER-associated SGs is gene-selective. Intriguingly, we also discovered that SG biogenesis is intimately linked to, and dependent upon, de novo transcription. These findings are significant because they suggest that SG biogenesis is restricted to newly exported mRNAs, is spatially organized on the ER membrane, and reveal divergent fates for mature vs. newly exported mRNAs during the cellular response to stress. By identifying molecular signals for the recruitment of mRNAs into SGs and determining how transcription and SG biogenesis are coupled, we expect that the candidate’s research will advance understanding of stress granule biology and its regulatory ro...