Intermolecular RNA-RNA interactions in stress granule regulation and disease - Project Summary Ribonucleoprotein (RNP) granules are membraneless organelles formed from RNA and RNA binding proteins (RBPs) through biomolecular condensation or liquid-liquid phase separation. Stress granules (SGs) are prototypical RNP granules, which form transiently under stresses such as arsenite exposure, UV damage, and viral infection. The unifying thread between these stresses is translation shutdown due to eiF2a phosphorylation, leading to rapid increases in the concentration of lowly bound cytoplasmic RNA, which coalesces with RBPs to form SGs. Chronic stress exposures or mutations in SG RBPs lead to constitutive SGs that behave more like aggregates than condensates. These stable SGs correlate with poor health outcomes such as aging, asthma, amyotrophic lateral sclerosis, frontotemporal dementia, muscular dystrophy, and cancer. Thus, understanding the mechanisms regulating SGs and their functional roles in the cell is paramount to treating disease and promoting extended health spans. Though SGs are known to involve extensive networks of protein-protein and protein-RNA interactions, it remains poorly understood how intermolecular RNA-RNA interactions contribute to SG organization and function. Therefore, the proposed research objectives focus on determining how the SG scaffolding protein G3BP1 facilitates the formation of intermolecular RNA-RNA interactions, what RNA elements compose these interactions, and how the cell mitigates stable RNA-RNA interactions during normal cell function of stress recovery to prevent progression into disease states. In this work, approaches using biochemical, transcriptomic, high-throughput genetic screens, and cancer cell progression models will 1) determine the biochemical features required for RNP granule scaffolding proteins to form intermolecular RNA-RNA interactions and their impacts on stress response, 2) characterize the RNA elements involved in forming stable RNP granule RNA networks, and 3) identify the cohort of proteins responsible for limiting intermolecular RNA-RNA interactions and their roles in preventing progression from healthy to disease states. In the K99 phase, mentorship from Dr. Roy Parker and Dr. Robin Dowell will provide valuable training in biochemical and high-throughput sequencing techniques required to determine the mechanisms and features driving RNA network formation within stress granules. Further support from Dr. Jennifer DeLuca will provide skills in working with cancer progression models to determine the functional effects of SG RNA network dysregulation. The training in biochemical, sequencing, computational, and cell-biological techniques during the K99 phase will be essential to the proposed research and develop valuable skills required for the independent R00 phase. Ultimately, the results from this proposal will advance our understanding of SGs and RNP granules generally by revealing the fundamental role of RNA-RNA interactions in granule organization and maintenance.
