Molecular mechanism underlying FUS regulation under transcriptional and DNA damage stress

PROJECT SUMMARY Fused in sarcoma (FUS) is an RNA binding protein (RBP) capable of undergoing phase separation in the nucleus and is crucial for proper cellular function. Mutations in FUS have been linked to several incurable age-dependent neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), leading to progressive dysfunction and loss of neurons. Aberrant phase separation in ALS/FTLD-linked FUS mutants disrupts essential homeostatic mechanisms such as DNA damage response (DDR) and transcription regulation. Despite initial studies linking FUS with both genome maintenance and RNA-related functions, FUS' specific molecular mechanism and potential implications in neurodegenerative diseases, such as ALS/FTLD, remain elusive. We hypothesize that FUS subcellular localization changes under transcription inhibition and DNA damage and is dependent on FUS-RNA interactions. We propose that abnormal RNA interactions induced by ALS/FTLD-linked FUS mutants perturb this dynamic localization process under stress conditions. Advanced microscopy and next-generation sequencing approaches will be used in our model SH- SY5Y cells to test this hypothesis. Aim 1 will dissect the cellular mechanism modulating FUS dynamic localization under DNA damage and transcription stress conditions using immunofluorescence and high-resolution microscopy. Aim 2 will elucidate the RNA-dependent interactions influencing FUS localization through single molecule RNA fluorescence in situ hybridization (smRNA-FISH) and RNA immunoprecipitation sequencing (RIP- seq) before, during, and after stress induction. For Aim 3, we will employ high-resolution microscopy and RIP- seq to investigate how aberrant FUS-RNA interactions in ALS/FTLD-linked mutations disrupt FUS localization under stress, thereby contributing to disease onset. Collectively, these aims will provide mechanistic insights into the functions of FUS under DNA damage and transcription stress, and how these functions may differ in ALS/FTLD-linked mutants, shedding light towards understanding other RBP mutations in age-dependent neurodegenerative diseases. My proposed research is strengthened by collaborations with experts in neurodegeneration, ALS/FTLD, RNA biology, phase separation, next-generation sequencing, and high- resolution microscopy (see support letters). The activities outlined in this proposal, including collaborations, professional development opportunities, engagement in scientific conferences, mentoring students, and improving scientific communication and critical thinking skills, will allow me to successfully complete my Ph.D. and prepare me for a competitive postdoctoral position, ultimately paving the way for a career in academia.