Determining subcellular coordination of Signal Recognition Particle assembly

About This Grant

Project Summary/Abstract: Protein targeting is essential for maintaining cellular organization and function across the tree of life. Signal Recognition Particle (SRP) is a ribonucleoprotein complex that plays an important role in this process, co- translationally targeting 30% of the proteome for membrane insertion or secretion. SRP recognizes the signal sequence of secretory proteins, pauses translation, and guides the ribosome-nascent chain complex to the endoplasmic reticulum. Errors in SRP-dependent protein targeting lead to protein aggregation and fitness defects, and are associated with developmental disorders, neurodegeneration, and cancer. Despite SRP’s important function, the mechanisms underlying its assembly remain poorly understood. Specifically, the factors involved in SRP assembly and how assembly is subcellularly coordinated remains largely unknown. Therefore, the goal of this proposal is to identify SRP assembly factors and characterize their mechanism in human cells. Structurally, SRP consists of six proteins and a 300-nucleotide noncoding RNA, 7SL. Using microscopy, we and others have observed that several of these components localize to nucleoli, which are biomolecular condensates that form through liquid-liquid phase separation and facilitate ribosome biogenesis. Based on these observations, I hypothesize that nucleoli concentrate factors that mediate 7SL maturation, which is necessary for SRP assembly. This will be tested through two specific aims. In Aim 1, I will identify 7SL RNA-binding proteins and investigate their role in SRP assembly and 7SL folding. In Aim 2, I will employ CRISPR screening to identify nucleolar SRP assembly factors and determine whether their condensation contributes to function. By testing whether nucleoli coordinate SRP assembly, this work will advance our understanding of biomolecular condensates and ribonucleoprotein biogenesis, revealing novel therapeutic targets for diseases that arise from protein mistargeting. Furthermore, this study constitutes the research arm of a comprehensive training plan that facilitates my development as a physician-scientist. Throughout this proposal, I will have mentored training in molecular biology research and gain the skills necessary to become an independent investigator, including experimental design, scientific writing, and presentation skills. This work will be conducted at the Omenn-Darling Bioengineering Institute at Princeton University, which has state-of-the-art microscopy, flow cytometry, and genomics equipment. My research training will be complemented with longitudinal clinical training in precision medicine and hematology-oncology at the Rutgers Cancer Institute. Together, my research and clinical training will provide a strong foundation for pursuing a career as an independent physician-scientist.