PROJECT SUMMARY Fused in sarcoma (FUS) is an abundant nuclear RNA binding protein that helps regulate nearly every level of RNA processing. FUS activity depends on its ability to specifically bind RNA, however it remains unknown how it achieves specificity given its affinity for a wide variety of cellular RNA sequences. A striking example of FUS specificity is on its own pre-mRNA where it binds exclusively near exon 7 to repress exon skipping, leading to nonsense mediated decay and FUS autoregulation. In neurological diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), FUS mislocalizes to the cytoplasm, incorporates into stress granules, and subsequently forms pathological inclusions. FUS misregulation in ALS/FTD causes widespread disruption of gene expression including FUS autoregulation, leading to overexpression of FUS and amplification of pathogenic aggregates. Although RNA binding is central to the physiological and pathological activities of FUS, the FUS:RNA interaction and its functional outcome remain poorly understood. This proposal seeks to investigate the FUS:RNA interaction across multiple scales to better understand FUS RNA-binding preferences and the role of RNA sequence and structure in defining FUS activity. Aim 1 will test FUS binding to a broad set of synthetic and biological RNA constructs including exon 7 from FUS mRNA. The FUS:RNA interaction will be characterized using EMSA, fluorescence anisotropy, single molecule FRET and NMR spectroscopy. This aim will also test the impact of ALS-associated FUS mutations on RNA binding. Aim 2 will test these RNAs in phase separation assays based on the fact that FUS readily phase separates into liquid droplets in an RNA-dependent manner. How different RNAs and FUS variants affect the size, number, shape, and fluidity of FUS droplets and how this corresponds to their molecular interactions determined in aim 1 will be observed. Finally, aim 3 will test the RNA dependence of FUS splicing activity in neuroblastoma and neuronal cells using an RT-PCR based minigene reporter assay and real-time single molecule imaging. Special attention will be given to FUS autoregulation due to its relevance to ALS/FTD. Together, these aims will help uncover the mechanism of FUS specificity and activity. The proposed research will primarily be carried out in the lab of Dr. Sua Myong with support and co-mentorship from Dr. Jiou Wang. Dr. Myong will provide expertise in RNA-protein biophysics and single molecule methods while Dr. Wang has expertise in neurobiology and neurodegeneration, specifically ALS/FTD. Between the sponsors, collaborators, and scientific community at Johns Hopkins the applicant will receive the mentorship, technical training, and access to resources and expertise necessary to accomplish the proposed project. Additionally, the proposal outlines many planned activities for career development including mentorship, teaching, and scientific communication. Overall, this ...