An Integrated Biochemical and Structural Approach to Delineating the Biology of EWSR1

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PROJECT SUMMARY Fundamental cellular functions such as transcription, RNA processing, and DNA damage repair are achieved through the correct spatial and temporal localization of biomolecular complexes containing dozens of different protein and nucleic acid species. RNA-binding proteins that contain low-complexity amino acid sequences are essential components of these complexes but also form pathological assemblies in neurodegenerative diseases and pediatric cancers. The function of these low complexity sequences in healthy and disease states remain poorly understood, partly because of the difficulty in obtaining high resolution structural information of the pro- teins participating in these assemblies. The RNA binding protein Ewing sarcoma breakpoint 1 (EWSR1) is mem- ber of a group of approximately 70 human RNA-binding proteins that contain intrinsically disordered low-com- plexity regions that are deficient in charged amino acids but contain a high proportion of aromatic residues. These low complexity regions self-associate, driving the assembly of dynamic clusters in a process commonly referred to as liquid-liquid phase separation. EWSR1 primarily functions in mRNA processing and maturation through the formation of dynamic, reversible complexes that provide a scaffold for, and promote the correct spatial location of the processing machinery. Mutations in the low-complexity region cause uncontrolled assem- bly of EWSR1 (and related proteins) forming pathological inclusions linked to the progression of amyotrophic lateral sclerosis, frontal temporal dementia and related neuropathies. Further, through chromosomal transloca- tions, the low-complexity domain of EWSR1 is joined to DNA-binding domains forming potent oncogenic fusions responsible for the development of pediatric sarcomas. There is a paucity of molecular structural information on the pathogenic function of EWSR1 and particularly how the low-complexity domain contributes to the oncogenic properties of EWSR1-fusions. Recent technological advances in NMR spectroscopy now enable detection and quantification of the dynamic, highly transient interactions that drive complex formation, thus providing the req- uisite tool for determining the structure and function of EWSR1. This project will employ advanced NMR spec- troscopic and other biophysical techniques, fluorescent and hydrodynamic methods, spectroscopic aggregation assays and microscopy in conjunction with biochemical and biological assays to: (1) determine the molecular events leading to EWSR1 self-assembly and biomolecular condensation; (2) define the structural details of how the low complexity domain contributes to normal and abnormal EWSR1 functions; and (3) determine the role of phase separation in the formation and stabilization of protein:DNA complexes involving the oncogenic EWS-FLI1 fusion protein. The results of our investigations will help advance our general understanding about macromolec- ular assembly of dynamic protein/nucleoprotein complexes formed by low-complexity proteins. Understanding the structural basis that defines their activity will guide the development of strategies to therapeutically target low-complexity proteins or their molecular partners.